Team:UESTC-China/Protocol
Pichia pastoris
1.1 Common media for GS115 screening and fermentation
1.1.1 YPD liquid medium
In the early stage of Pichia pastoris GS115 amplification, we use YPD liquid medium.
1% yeast extract
2% peptone
2% glucose
Glucose needs to be sterilized separately and mixed after sterilization.
2% peptone
2% glucose
1.1.2 Screening medium YPDZ
In order to screen the successfully recombined Pichia pastoris GS115 after electroporation transformation, we use YPDZ.
1% yeast extract
2% peptone
2% glucose
1.5%-2% agar powder
100ug/ml zeocin
Glucose needs to be sterilized separately and mixed after sterilization.
2% peptone
2% glucose
1.5%-2% agar powder
100ug/ml zeocin
1.1.3 MDH medium
MDH medium is not as rich in nutrients as YPD, and is used for the separation and purification of yeast.
13.4g/L YNB
20g/L glucose
0.4mg/L biotin
0.004% histidine
1.5%-2% agar powder
Glucose is sterilized separately, biotin and histidine are sterilized with a filter membrane and then added to the medium and mixed.
20g/L glucose
0.4mg/L biotin
0.004% histidine
1.5%-2% agar powder
1.1.4 BMGY medium
BMGY medium is a complex buffer medium containing glycerol.
1% yeast powder
2% peptone
13.4g/L YNB
1% glycerol
It is usually used for expanded culture in the early stage of fermentation.
2% peptone
13.4g/L YNB
1% glycerol
1.1.5 Modified YPD medium
Modified YPD medium is the standard medium for pichia pastoris to propagate.
1% yeast extract
2% peptone
6%NaCl
2% glycerin
It is usually for yeast expression.
2% peptone
6%NaCl
2% glycerin
1.2 Recovery, validation and conservation of Pichia pastoris
Steps：
1. Take Pichia pastoris GS115 preserved at -80℃.
2. After dipping with an inoculating loop, inoculate on MDH and MD plates by streaking. Since GS115 is a histidine-deficient strain, it cannot grow on MD plates. And can grow on MDH plates containing histidine.
3. Compare the growth of yeast on the two plates to verify.
4. Pick a single colony from the MDH plate and inoculate it in YPD liquid culture.
5. Cultivate at 30℃, 250rpm, when the OD value is 1.5.
6. Take 1ml of bacteria liquid into 2ml of sterilized EP tube, add 1ml of 50% glycerin, and store at -20℃.
2. After dipping with an inoculating loop, inoculate on MDH and MD plates by streaking. Since GS115 is a histidine-deficient strain, it cannot grow on MD plates. And can grow on MDH plates containing histidine.
3. Compare the growth of yeast on the two plates to verify.
4. Pick a single colony from the MDH plate and inoculate it in YPD liquid culture.
5. Cultivate at 30℃, 250rpm, when the OD value is 1.5.
6. Take 1ml of bacteria liquid into 2ml of sterilized EP tube, add 1ml of 50% glycerin, and store at -20℃.
1.3 Electrotransformation of Pichia pastoris
1.3.1 Preparation of linearized DNA
Steps：
1. The 50μL system of linearization reaction is: (the enzyme is added at the end)
3. Agarose gel electrophoresis to verify complete linearization.
Restriction digestion verification system 6ul Restriction digestion verification control system 6ul:
Notes:
① 10xL Buffer 5 uL (If only the restriction enzyme digestion verification is performed, a colored buffer can be added; if other operations are to be performed, a transparent buffer can be added).
②Recombinant plasmid 5-10ug (In order to make the total volume not exceed 50ul, the plasmid concentration is generally above 140ng/ul and leave at least 2ul of the undigested plasmid and run the electrophoresis verification together with the digested plasmid).
③Enzyme: digest 0.5-1ul overnight.
④ddH2O allocated to 50ul.
2. After mixing thoroughly, use a 37℃ water bath (the time can be longer to ensure sufficient digestion and then take 2μL of the digested product and perform agarose gel electrophoresis to observe whether it is completely linearized). ②Recombinant plasmid 5-10ug (In order to make the total volume not exceed 50ul, the plasmid concentration is generally above 140ng/ul and leave at least 2ul of the undigested plasmid and run the electrophoresis verification together with the digested plasmid).
③Enzyme: digest 0.5-1ul overnight.
④ddH2O allocated to 50ul.
3. Agarose gel electrophoresis to verify complete linearization.
Restriction digestion verification system 6ul Restriction digestion verification control system 6ul:
3ul ddH2O
2ul plasmid after digestion
1ul Orange Buffer
Compare the two to see if the linearization is complete. 2ul plasmid after digestion
1ul Orange Buffer
1. Selected linearizing enzyme must not have a digestion check point in the target gene.
2. Enzymatic digestion time must be sufficient to linearize most plasmids.
3. Enzymes used for single digestion of six plasmids:
2. Enzymatic digestion time must be sufficient to linearize most plasmids.
3. Enzymes used for single digestion of six plasmids:
①EG1 EGl7 XynB - - Bgl
②LipLac & Scaffold - - Avr
③SdbA - - BspH
②LipLac & Scaffold - - Avr
③SdbA - - BspH
1.3.2 Ethanol precipitation of linearized DNA
Steps：
1. DNA precipitation: place the system in a 65℃ water bath for 20 minutes after digestion. Anhydrous ethanol, 80% ethanol pre-cooling.
2. Add 1/10 (system after enzyme digestion) volume of 3M pH5.2 NaAC and 2.5 times volume of pre-cooled absolute ethanol, mix well, and place at -20°C for more than 35 minutes.
3. Centrifuge at 12000rpm for 15min, discard the supernatant.
4. Wash the pellet with about 300-400ul pre-cooled 80% ethanol (resuspend), centrifuge at 12000rpm for 10min.
5. Aspirate the supernatant (ethanol) and air dry for some time, (for example, 10 minutes, until there is no ethanol in the tube).
6. Resuspend in 10ul ddH2O.
Note:
2. Add 1/10 (system after enzyme digestion) volume of 3M pH5.2 NaAC and 2.5 times volume of pre-cooled absolute ethanol, mix well, and place at -20°C for more than 35 minutes.
3. Centrifuge at 12000rpm for 15min, discard the supernatant.
4. Wash the pellet with about 300-400ul pre-cooled 80% ethanol (resuspend), centrifuge at 12000rpm for 10min.
5. Aspirate the supernatant (ethanol) and air dry for some time, (for example, 10 minutes, until there is no ethanol in the tube).
6. Resuspend in 10ul ddH2O.
The precipitate may not be seen, and the DNA is stuck on the wall of the EP tube.
1.3.3 Preparation of Pichia pastoris
Steps：
1. Pick a single yeast colony and inoculate it into a 50ml Erlenmeyer flask containing 5ml YPD medium at 30°C, 250-300 revolutions/min, and cultivate overnight.
2. Take 100~500μl (one-thousandth) of the culture and inoculate it into a 200ml triangle shaker containing 100ml (reagents generally use 110-120ml liquid medium, and the excess is used for -80°C preservation and OD measurement) of fresh medium. In the bottle, incubate overnight at 30°C and 250-300rpm until the OD600 reaches 1.3-1.5 (note the record); pre-cool the sterile water and sorbitol solution.
3. Centrifuge the cell culture at 4°C and 7000 rpm for 6 min, and discard the supernatant.
Resuspend the bacterial pellet with 50 mL of ice-cold sterile water.
4. Centrifuge according to step 3, and resuspend the bacterial pellet with 25ml of ice-cold sterile water.
5. Centrifuge according to step 3, and resuspend the bacterial pellet with 5 ml ice-cold 1 M sorbitol solution.
6. Centrifuge according to step 3, and resuspend the bacterial pellet with 160ul ice-cold 1M sorbitol solution, the final volume of which is about 240ul.
7. (Optional) It can be divided into 80μl aliquots and frozen at -80℃ and used within 2 weeks, but it may affect its conversion efficiency.
2. Take 100~500μl (one-thousandth) of the culture and inoculate it into a 200ml triangle shaker containing 100ml (reagents generally use 110-120ml liquid medium, and the excess is used for -80°C preservation and OD measurement) of fresh medium. In the bottle, incubate overnight at 30°C and 250-300rpm until the OD600 reaches 1.3-1.5 (note the record); pre-cool the sterile water and sorbitol solution.
3. Centrifuge the cell culture at 4°C and 7000 rpm for 6 min, and discard the supernatant.
Resuspend the bacterial pellet with 50 mL of ice-cold sterile water.
4. Centrifuge according to step 3, and resuspend the bacterial pellet with 25ml of ice-cold sterile water.
5. Centrifuge according to step 3, and resuspend the bacterial pellet with 5 ml ice-cold 1 M sorbitol solution.
6. Centrifuge according to step 3, and resuspend the bacterial pellet with 160ul ice-cold 1M sorbitol solution, the final volume of which is about 240ul.
7. (Optional) It can be divided into 80μl aliquots and frozen at -80℃ and used within 2 weeks, but it may affect its conversion efficiency.
1.3.4 Electrotransformation
Pre-cooled 2mm electro-rotor cup, sorbitol solution
Steps：
Steps：
1. 10ul + 80ul competent Pichia pastoris solution with linearized DNA (mix in the EP tube first and then ice bath), transfer to a 2mm electrotransformation cup (added to the gap of the electroporation cup) that has been pre-cooled for at least 5 minutes.
Take out the dry electrode (the voltage is extremely high to avoid explosion).
2. Electric shock: (parameters have been set, no need to change) select fungus of igem or protols; parameter setting: voltage 1.5kV; capacitance 25μF; resistance 200-400Ω. The shock time is 5+-msec.
3. After the electric shock is finished, immediately add 1ml of 4℃ pre-cooled 1M sorbitol solution.
5. Spread the bacterial suspension gradient (200ul) on the YPDZ plate. If the bacterial concentration is high, coat multiple plates; place the plate at 30°C and incubate until a single colony appears, about 1-2 days.
Note:
2. Electric shock: (parameters have been set, no need to change) select fungus of igem or protols; parameter setting: voltage 1.5kV; capacitance 25μF; resistance 200-400Ω. The shock time is 5+-msec.
3. After the electric shock is finished, immediately add 1ml of 4℃ pre-cooled 1M sorbitol solution.
Mix gently with a pipette tip and transfer to a 1.5ml EP tube; do not shake!
4. Place the EP tube in a thermostat at 30°C and incubate for 1-2h. Don't shake it! 5. Spread the bacterial suspension gradient (200ul) on the YPDZ plate. If the bacterial concentration is high, coat multiple plates; place the plate at 30°C and incubate until a single colony appears, about 1-2 days.
1. Be careful to wipe the metal surface clean during electric shock, otherwise it may explode.
2. When inserting the electro-rotor, pay attention to the electrode surface and the metal surface of the electro-rotor.
3. Pay attention to "upside down" when placing the plate to prevent condensation from dripping into the culture medium.
2. When inserting the electro-rotor, pay attention to the electrode surface and the metal surface of the electro-rotor.
3. Pay attention to "upside down" when placing the plate to prevent condensation from dripping into the culture medium.
1.4 Colony PCR
Steps：
1. Prepare a 0.2ml centrifuge tube and add 20ul 0.02mol/L NaOH.
2. Use a toothpick to pick some of the colonies in the centrifuge tube.
3. The remaining points on the toothpicks are used as seeds on YPDS.
4. Set the centrifuge tube to 95 degrees Celsius, heat it for 5 minutes and take it out.
5. Do not need centrifugation, directly take 3-5ul as PCR template.
6. PCR system configuration (Qingke T3 Mix)
Note:
2. Use a toothpick to pick some of the colonies in the centrifuge tube.
3. The remaining points on the toothpicks are used as seeds on YPDS.
4. Set the centrifuge tube to 95 degrees Celsius, heat it for 5 minutes and take it out.
5. Do not need centrifugation, directly take 3-5ul as PCR template.
6. PCR system configuration (Qingke T3 Mix)
Total system 25ul
T3 Mix 18ul
Primer F 1ul
Primer R 1ul
DNA template 5ul
T3 Mix 18ul
Primer F 1ul
Primer R 1ul
DNA template 5ul
DNA template concentration should not be too high. If it is too high, add less DNA template, and use ddH2O to make up the rest.
1.5 Screening of recombinant in Pichia pastoris GS115 after electric transformation
In order to obtain multiple copies of transformants, the transformed single colonies shall be screened by G418.
Steps:
Steps:
1. Take a 96-well cell culture plate and add 200μL YPD to each well.
2. Use a sterile toothpick to pick a single colony (His+transformant) from the MD plate, stir and resuspend in the well which corresponds to a colony.
3. Cultivate at 30°C for two days.
4. Take the second 96-well cell culture plate, add 190μLYPD to each well, and then add 10μL of the bacterial solution from the first 96-well cell culture plate to each well, cover the lid and cultivate overnight at 30°C.
5. Repeat step 4. (Note: Continuous growth and passage can make the cell density of each transformant reach the same).
6. Resuspend the bacterial solution with a multi-channel gun, and take 1μL of bacterial solution from each well to grow on the YPD plate with a G418 concentration of 1.75mg/mL.
7. Cultivate and observe at 30°C.
2. Use a sterile toothpick to pick a single colony (His+transformant) from the MD plate, stir and resuspend in the well which corresponds to a colony.
3. Cultivate at 30°C for two days.
4. Take the second 96-well cell culture plate, add 190μLYPD to each well, and then add 10μL of the bacterial solution from the first 96-well cell culture plate to each well, cover the lid and cultivate overnight at 30°C.
5. Repeat step 4. (Note: Continuous growth and passage can make the cell density of each transformant reach the same).
6. Resuspend the bacterial solution with a multi-channel gun, and take 1μL of bacterial solution from each well to grow on the YPD plate with a G418 concentration of 1.75mg/mL.
7. Cultivate and observe at 30°C.
1.6 Pichia pastoris protein expression
Steps：
1. Cultivate the verified transformants in the 250ml triangular flask containing 25mL BMGY medium at 28℃/200 rpm until OD600 equals 2-6(usually 16-18h).
2. Centrifuge at 6000g for 5min at room temperature, collect the bacteria, and resuspend the bacteria with 50mLBMMY to make OD600 = about 1.0.
3. Put the bacterial solution obtained in step 2 in a 500mL shake flask, seal it with cheesecloth, and place it on a shaker at 28-30°C/200 rpm to continue culturing.
4. Take 1 mL of bacterial liquid samples at different time points at 0, 24, 48, 72, 96 and centrifuge them in a 1.5 mL EP tube at maximum speed for 2 to 3 minutes, and collect the supernatant and bacterial cells respectively.
5. Detect protein concentration (Bradford method) and enzyme activity in the supernatant.
2. Centrifuge at 6000g for 5min at room temperature, collect the bacteria, and resuspend the bacteria with 50mLBMMY to make OD600 = about 1.0.
3. Put the bacterial solution obtained in step 2 in a 500mL shake flask, seal it with cheesecloth, and place it on a shaker at 28-30°C/200 rpm to continue culturing.
4. Take 1 mL of bacterial liquid samples at different time points at 0, 24, 48, 72, 96 and centrifuge them in a 1.5 mL EP tube at maximum speed for 2 to 3 minutes, and collect the supernatant and bacterial cells respectively.
5. Detect protein concentration (Bradford method) and enzyme activity in the supernatant.
1.7 Screening and identification of transformed clones
1.7.1 yeast transformant zeocin screening
Steps：
1. In order to obtain a multi-copy transformant, zeocin screening was performed on the single colony obtained by transformation. 20 mL YPD was prepared, zeocin with a final concentration of 100 ug/ml was added, and plates were prepared.
2. Take 96-well cell culture plates and add 200 μL YPD per well.
3. A single colony (zeocin transformant) was picked from the MDHS plate with a sterilized toothpick, stirred and resuspended in the hole and each hole corresponds to one colony; incubated at 30 ℃ for two day.
4. Take the second 96-well cell culture plate, add 190 μL MDHZ to each well, and then add 10 μL of the bacterial solution of the first 96-well cell culture plate to each well, cover and culture at 30 ℃ overnight.
5. Repeat the previous step. (Note: Continuous growth and passage can make the cell density of each transformant consistent).
6. Resuspend the bacterial solution with a multi-channel gun, take 1 μL of bacterial solution from each well and grow on a YPD plate with a zeocin concentration of 100ug/mL.
7. 30 ℃ culture and observation.
2. Take 96-well cell culture plates and add 200 μL YPD per well.
3. A single colony (zeocin transformant) was picked from the MDHS plate with a sterilized toothpick, stirred and resuspended in the hole and each hole corresponds to one colony; incubated at 30 ℃ for two day.
4. Take the second 96-well cell culture plate, add 190 μL MDHZ to each well, and then add 10 μL of the bacterial solution of the first 96-well cell culture plate to each well, cover and culture at 30 ℃ overnight.
5. Repeat the previous step. (Note: Continuous growth and passage can make the cell density of each transformant consistent).
6. Resuspend the bacterial solution with a multi-channel gun, take 1 μL of bacterial solution from each well and grow on a YPD plate with a zeocin concentration of 100ug/mL.
7. 30 ℃ culture and observation.
1.7.2 PCR verification of yeast transformers
The step of alkaline lysis method is to put the selected bacteria (the bacterial solution is 1 ul, and the colony is to make the lysis system not cloudy) into 20ul 0.02mol NaoH, place it in the following PCR instrument at 97 ℃ for 10 min, and then take
5ul and add it to the prepared 20ulPCR system (18ul supermix + 1ulR primer + 1ulF primer).
Steps:
Steps:
1. PCR verification will begin when the size of the bacteria growing on YPD after screening on the 96-well plate is found to be appropriate. At this time, the colony size of the screened bacteria indicates the number of multiple copies. Theoretically,
the larger the colony grows, the more copies it has, and the PCR band should be brighter.
2. Pick the yeast transformers with a toothpick and dip them in a sterilized amplification tube, then place the toothpick in a sterilized 1.5 mL eppendorf tube for storage.
3. Prepare PCR system:
Notes：
2. Pick the yeast transformers with a toothpick and dip them in a sterilized amplification tube, then place the toothpick in a sterilized 1.5 mL eppendorf tube for storage.
3. Prepare PCR system:
①1.1×T3 Super PCR Mix 18-22ul
②10 uM Primer F 1ul
③10 uM Primer R 1ul
④Template DNA
⑤ddH2O
⑥Guarantee that the total PCR system should be 25ul and mixed evenly
4. PCR reaction program setup：
②10 uM Primer F 1ul
③10 uM Primer R 1ul
④Template DNA
⑤ddH2O
⑥Guarantee that the total PCR system should be 25ul and mixed evenly
①97 ℃ 10min (broken wall)
②94 ℃, 5min (initial chain solution)
③94 ℃ 30sec (unchain)
④56 ℃ 30sec (annealed)
⑤72 ℃ 2min (extended)
⑥Go to step 3 42cycles
⑦72 ℃ 10min (final extension)
⑧4℃ hold
②94 ℃, 5min (initial chain solution)
③94 ℃ 30sec (unchain)
④56 ℃ 30sec (annealed)
⑤72 ℃ 2min (extended)
⑥Go to step 3 42cycles
⑦72 ℃ 10min (final extension)
⑧4℃ hold
1. The plasmid containing the foreign gene was used as the template as the positive control. After the amplification was completed, 1.2% agarose gel electrophoresis was performed.
2. The result should read:
2. The result should read:
(1)Non-specific primer pGAPz forward + 3 'AOX1: two bands:
①500Bp yeast internal reference
②X + 500bp destination strip
③EG1:4460+500=4950bp
④EGL7:4508+500=5058bp
⑤xynB:3974+500=4474bp
⑥scaffold:5582+500=6082bp
⑦sdbA:4700+500=5200bp
⑧Liplac:6386+500=6886bp
(2)Specific primers: one-day band:
②X + 500bp destination strip
③EG1:4460+500=4950bp
④EGL7:4508+500=5058bp
⑤xynB:3974+500=4474bp
⑥scaffold:5582+500=6082bp
⑦sdbA:4700+500=5200bp
⑧Liplac:6386+500=6886bp
①EG1: 1072bp
②EGL7: 694bp
③xynB: 662bp
④scaffold: 1901bp
⑤sdbA: 707bp
⑥Liplac: 861bp
②EGL7: 694bp
③xynB: 662bp
④scaffold: 1901bp
⑤sdbA: 707bp
⑥Liplac: 861bp
E.coil
2.1 Plasmid Mini Preparation
Materials:
1.Buffer S1
2.Buffer S2
3.Buffer S3
4.Buffer W1
5.Buffer W2
6.UP water
2.Buffer S2
3.Buffer S3
4.Buffer W1
5.Buffer W2
6.UP water
Steps:
1. Centrifuge 1-4ml overnight cultures at 12,000 rpm for 1 mins, and discard the supernatant.
2. Resuspend the pellet with 250μl Buffer S1.(before this step, ensure RNaseA has been all added into Buffer S1 and there is no white sediment on the bottom of buffer)
3. Add 250μl Buffer S2 and mix by softly inverting 10 times, then wait 2-3mins. This step should not exceed 5 mins.
4. Add 350 µl of Buffer S3 and mix by softly inverting 6-8 times. This step would observe white precipitate. Centrifuge at 12,000 rpm for 10 mins.
5. Transfer the supernatant into a QIAprep 2.0 spin column. Centrifuge at 12,000 rpm for 1 min and discard the filtrate.
6. Add 500µl of Buffer W1 and centrifuge at 12,000 rpm for 1 min. Discard wash-through.
7. Wash filter membrane twice by adding 700µl of Buffer W2 and centrifuge at 12,000 rpm for 1 min. Discard wash-through.(before this step, ensure ethanol has been added into Buffer S2)
8. Open the tube and centrifuge at 12,000 rpm for 1 min. Then wait until ethanol has volatilized.
9. For elution, add 80-100µl of 65℃ UP water to the center of the QIAquick membrane without touching it. Let stand at room temperature for 1 min then centrifuge at 12,000 rpm for 1 min. Do not throw the flow-through as it contains the purified PCR product.
10. Determine purified plasmid concentration using nanodrop.
2. Resuspend the pellet with 250μl Buffer S1.(before this step, ensure RNaseA has been all added into Buffer S1 and there is no white sediment on the bottom of buffer)
3. Add 250μl Buffer S2 and mix by softly inverting 10 times, then wait 2-3mins. This step should not exceed 5 mins.
4. Add 350 µl of Buffer S3 and mix by softly inverting 6-8 times. This step would observe white precipitate. Centrifuge at 12,000 rpm for 10 mins.
5. Transfer the supernatant into a QIAprep 2.0 spin column. Centrifuge at 12,000 rpm for 1 min and discard the filtrate.
6. Add 500µl of Buffer W1 and centrifuge at 12,000 rpm for 1 min. Discard wash-through.
7. Wash filter membrane twice by adding 700µl of Buffer W2 and centrifuge at 12,000 rpm for 1 min. Discard wash-through.(before this step, ensure ethanol has been added into Buffer S2)
8. Open the tube and centrifuge at 12,000 rpm for 1 min. Then wait until ethanol has volatilized.
9. For elution, add 80-100µl of 65℃ UP water to the center of the QIAquick membrane without touching it. Let stand at room temperature for 1 min then centrifuge at 12,000 rpm for 1 min. Do not throw the flow-through as it contains the purified PCR product.
10. Determine purified plasmid concentration using nanodrop.
2.2 Agarose gel
Materials:
1. Agarose
2.1×TAE buffer
3.Gel red
2.1×TAE buffer
3.Gel red
Steps:
1. Prepare 1.2% agarose gel by adding agarose in 1×TE buffer, heating until agarose melt and adding 0.01% gel red.
2. Mix and pour into the mould.
3. Prepare the sample by mixing result with 6×Orange Buffer.
4. Add mixture and DNA marker into different holes.
5. Running 20mins at 130V.
2. Mix and pour into the mould.
3. Prepare the sample by mixing result with 6×Orange Buffer.
4. Add mixture and DNA marker into different holes.
5. Running 20mins at 130V.
2.3 PCR purification （AxyGEN)
Materials：
1. Buffer DE-A
2. Buffer DE-B
3. Buffer W1
4. Buffer W2 concentrate
5. UP water
6. QIAquick column
2. Buffer DE-B
3. Buffer W1
4. Buffer W2 concentrate
5. UP water
6. QIAquick column
Steps:
1. Excise the DNA fragment from agarose gel with a clean scalpel and place it in an eppendorf tube. Weigh the gel slice and count its volume(100mg=100μl). Set it as 1 unit.
2. Add 3 units Buffer DE-A and bath at 75℃ with 1 volumes gel. Mix until the gel melts( about 6-8 mins).
3. Add 1.5 units of Buffer DE-B and mix completely. The colour of the mixture will turn yellow.
4. Place a QIAquick column in a provided 2 ml collection tube.
5. Transfer the sample to the QIAquick column and centrifuge at 12,000 rpm for 1 min to bind DNA. Discard flow-through.
6. Add 500μl buffer W1 to the column and centrifuge at 12,000 rpm for 30s. Discard flow-through.
7. For washing, add 700 µl buffer W2 to the column and centrifuge at 12,000 rpm for 30s then discard the flow-through.
8. Centrifuge again at 12,000 rpm for 1 min to remove any residual buffer.
9. Place the QIAquick column in a clean 1.5 ml eppendorf tube.
10. For elution, add 25-30 µl UP water to the center of the QIAquick membrane without touching it. Let stand at room temperature for 1 mins then centrifuge at 12,000 rpm for 1 min. Do not throw the flow-through as it contains the purified PCR product.
11. Use the nanodrop to determine concentrations of the PCR purification product.
2. Add 3 units Buffer DE-A and bath at 75℃ with 1 volumes gel. Mix until the gel melts( about 6-8 mins).
3. Add 1.5 units of Buffer DE-B and mix completely. The colour of the mixture will turn yellow.
4. Place a QIAquick column in a provided 2 ml collection tube.
5. Transfer the sample to the QIAquick column and centrifuge at 12,000 rpm for 1 min to bind DNA. Discard flow-through.
6. Add 500μl buffer W1 to the column and centrifuge at 12,000 rpm for 30s. Discard flow-through.
7. For washing, add 700 µl buffer W2 to the column and centrifuge at 12,000 rpm for 30s then discard the flow-through.
8. Centrifuge again at 12,000 rpm for 1 min to remove any residual buffer.
9. Place the QIAquick column in a clean 1.5 ml eppendorf tube.
10. For elution, add 25-30 µl UP water to the center of the QIAquick membrane without touching it. Let stand at room temperature for 1 mins then centrifuge at 12,000 rpm for 1 min. Do not throw the flow-through as it contains the purified PCR product.
11. Use the nanodrop to determine concentrations of the PCR purification product.
Notes:
1. Add ethanol (100%) to buffer W2 concentrate and set waterbath at 75℃ in advance. If there is white sediment on the bottom of Buffer DE-B, heat at 70℃ until it disappears and cools down to RT)
2.4 Double enzyme digestion system
Double enzyme digestion system preparation:
1. DNA:30μL
2. Enzyme 1:2.5 μL
3. Enzyme 2:2.5 μL
4. 10×Qcut:5 μL
5. ddH2O:10 μL
6.37℃ water bath insulation overnight.
2. Enzyme 1:2.5 μL
3. Enzyme 2:2.5 μL
4. 10×Qcut:5 μL
5. ddH2O:10 μL
6.37℃ water bath insulation overnight.
2.5 E.coil transformation
2.5.1 Chemically competent cells
Steps:
1. Grow overnight culture from single colony or competent cells aliquot.
2. Inoculate 1:100 in the desired volume of LB.
3. Grow to OD 600 ~0.35 (important that it is close to 0.35, 0.3-0.4 is good).
4. Chill on ice for 20 min (it can chill for a 1-2 hours).
5. Centrifuge at 8000g for 8 minutes in Falcon tubes, decant supernatant.
6. Resuspend by pipetting gently in 1/10 (of the volume of LB from step 2) of ice-cold TSS.
2. Inoculate 1:100 in the desired volume of LB.
3. Grow to OD 600 ~0.35 (important that it is close to 0.35, 0.3-0.4 is good).
4. Chill on ice for 20 min (it can chill for a 1-2 hours).
5. Centrifuge at 8000g for 8 minutes in Falcon tubes, decant supernatant.
6. Resuspend by pipetting gently in 1/10 (of the volume of LB from step 2) of ice-cold TSS.
Notes:
1. It can take a while to break up the pellet (you can use a 25 mL pipette and scrape the pellet off the side of the tube before pipetting).
3. Always do a transformation positive control using a plasmid that you know is efficiently taken up by competent cells to confirm competency
4. Always do a negative control transformation (add untransformed cells to a plate with antibiotics) to confirm that you don't have an antibiotic-resistant contaminant strain in your competent cell stock(This happens from time to time and can cause lots of agony!)
①10 g PEG 3350
②5 mL DMSO
③2 mL 1 M MgCl2
④LB to 100 mL
2. Freeze 100 µL aliquots in liquid nitrogen & store at -80ºC in 1,5mL Eppendorf tubes. ②5 mL DMSO
③2 mL 1 M MgCl2
④LB to 100 mL
3. Always do a transformation positive control using a plasmid that you know is efficiently taken up by competent cells to confirm competency
4. Always do a negative control transformation (add untransformed cells to a plate with antibiotics) to confirm that you don't have an antibiotic-resistant contaminant strain in your competent cell stock(This happens from time to time and can cause lots of agony!)
2.5.2 Transformation
Steps:
1. Put a tube of BL21 Competent E. coli cells on ice for 10 minutes.
2. Add 2µL of plasmid DNA to the cell mixture.
3. Mix by flicking the tube.
4. Place the tube on ice for 30 minutes.
5. Heat shock at 42ºC for exactly 10 seconds.
6. Place on ice for 5 minutes.
7. Add 950µL of SOC medium at room temperature.
8. Incubate at 37ºC with shaking for 1 hour.
2. Add 2µL of plasmid DNA to the cell mixture.
3. Mix by flicking the tube.
4. Place the tube on ice for 30 minutes.
5. Heat shock at 42ºC for exactly 10 seconds.
6. Place on ice for 5 minutes.
7. Add 950µL of SOC medium at room temperature.
8. Incubate at 37ºC with shaking for 1 hour.
Notes:
1. Not at the thermoblock, use a shacking incubator.
2. To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.
3. Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.
2. To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.
3. Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.
2.6 Escherichia coli culture medium preparation
We use low salt LB medium to culture E. Coli.
Low salt LB medium formulation：
Low salt LB medium formulation：
1. 10g tryptone
2. 5g NaCl
3. 5g yeast extract
4. Add distilled water to 1000ml
5. Adjust pH to 7.5 with 1M NaOH
6. If LB solid medium is prepared, add 15g agar per 1000ml
7. 121℃，20min in autoclave
8. Store at 4℃
2. 5g NaCl
3. 5g yeast extract
4. Add distilled water to 1000ml
5. Adjust pH to 7.5 with 1M NaOH
6. If LB solid medium is prepared, add 15g agar per 1000ml
7. 121℃，20min in autoclave
8. Store at 4℃
Protein detection
3.1 Western Blot
Polyacrylamide gel electrophoresis is a method of protein or nucleic acid separation using polyacrylamide gel as supporting medium. Polyacrylamide gel is a three dimensional network structure gel formed by the polymerization of acrylamide monomer and
crosslinking agent N, N- methylene bisacrylamide under the action of catalyst. By changing the ratio of monomer concentration to crosslinker, gels with different pore sizes can be obtained for separating substances with different molecular
weights.
SDS, or sodium dodecyl sulfate, is an anionic surfactant, which can combine with protein in a certain proportion to form a SDS-protein complex. At this time, the protein has a large amount of negative charge, and far more than its original charge, so that the charge difference between the natural protein molecules is reduced or eliminated. At the same time, the structure of proteins becomes loose and the shape tends to be uniform under the action of SDS, so the differences in the electrophoretic mobility of various SDS-protein complexes during electrophoresis only depend on the molecular weight of the protein.
Materials：
SDS, or sodium dodecyl sulfate, is an anionic surfactant, which can combine with protein in a certain proportion to form a SDS-protein complex. At this time, the protein has a large amount of negative charge, and far more than its original charge, so that the charge difference between the natural protein molecules is reduced or eliminated. At the same time, the structure of proteins becomes loose and the shape tends to be uniform under the action of SDS, so the differences in the electrophoretic mobility of various SDS-protein complexes during electrophoresis only depend on the molecular weight of the protein.
Materials：
1.30% gel storage solution: ACR 30g + Bis 0.8g was dissolved in 100 mL deionized water, filtered into brown bottles with filter paper, and stored at 4℃ to avoid light.
2.1.5mol/L Tris-buffer,pH8.8; 1 mol/L Tris HCl, pH6.8.
3.10% SDS: dissolve 10 grams of SDS in 100mL deionized water and store at room temperature.
4.TEMED: 10% concentration, 20 mL, 4℃ storage.
5.10% AP: 10 mL, freshly prepared, divided into 1.5mL centrifuge tube, stored at -20℃ for later use.
6.4X protein loading buffer:
2.1.5mol/L Tris-buffer,pH8.8; 1 mol/L Tris HCl, pH6.8.
3.10% SDS: dissolve 10 grams of SDS in 100mL deionized water and store at room temperature.
4.TEMED: 10% concentration, 20 mL, 4℃ storage.
5.10% AP: 10 mL, freshly prepared, divided into 1.5mL centrifuge tube, stored at -20℃ for later use.
6.4X protein loading buffer:
Tris (200mM, pH6.8) 10mL of 1M Tris (pH6.8)
SDS(8%) 20ml of 20% SDS
Bromphenol blue (0.4%), 0.2 mg
Glycerol (40%) 20ml
Add water up to 50ml. Stored at RT
SDS(8%) 20ml of 20% SDS
Bromphenol blue (0.4%), 0.2 mg
Glycerol (40%) 20ml
Add water up to 50ml. Stored at RT
7. Electrophoretic buffer:
Tris 3 g
Glycine 18.8 g
20% SDS 5 ml
Add H2O up to 1L
Stored at RT.
Glycine 18.8 g
20% SDS 5 ml
Add H2O up to 1L
Stored at RT.
8. Ponceau S staining solution: 10X Ponceau S solution was diluted to 1X by adding H2O.
9. Western transfer buffer:
9. Western transfer buffer:
Tris 3 g
Glycine 18.8 g
methonal 200 ml Add H2O up to 1L
Cool at 4℃.
Glycine 18.8 g
methonal 200 ml Add H2O up to 1L
Cool at 4℃.
10. 10XTBST:
Tris 24g
NaCl 80g
Adjust pH to 7.5 and add H2O up to 1L. Stored at RT.
NaCl 80g
Adjust pH to 7.5 and add H2O up to 1L. Stored at RT.
11. Protein blocking solution: 5% milk in TBST
12. Nitrocellulose membrane, primary antibody, secondary antibody, color substrate solution
12. Nitrocellulose membrane, primary antibody, secondary antibody, color substrate solution
Equipment：
Trace pipetting device, 4℃ / - 20℃ freezer, ice machine, desktop frozen high-speed centrifuge, multifunctional enzyme mark all wavelengths, gel imaging analysis system, protein electrophoresis, electrophoresis system capillary, transblot system, chest, transfer protein membrane system, automatic chemiluminescence image analysis system, high pressure sterilization pot, constant temperature electric heating oven, magnetic stirrer, decoloring shaking table.
Trace pipetting device, 4℃ / - 20℃ freezer, ice machine, desktop frozen high-speed centrifuge, multifunctional enzyme mark all wavelengths, gel imaging analysis system, protein electrophoresis, electrophoresis system capillary, transblot system, chest, transfer protein membrane system, automatic chemiluminescence image analysis system, high pressure sterilization pot, constant temperature electric heating oven, magnetic stirrer, decoloring shaking table.
3.1.1 Preparation of polyacrylamide gel:
Steps：
Preparation of concentrated gel solution. After mixing, it is injected into the top of the separating glue and inserted into the comb to avoid the appearance of bubbles.
1. Installation of the plastic plate model: wash the glass plate, air dry, and spare. When making glue, choose the right glass plate and assemble the plastic plate model.
2. The separation glue solution (20ml, available for two plates) is prepared, and the mixture is shaken gently by hand. The mixture is carefully injected into the gap of the prepared glass plates, leaving enough space (~ 2.5cm) for the concentrated glue. The 0.5ml deionized water (or isopropanol) is gently added to the top layer to cover it, so as to prevent the inhibition of the condensation by oxygen in the air. It can be seen that there is an interface between the water and the gel when the water is first added, then it gradually disappears, and then the interface appears again soon, which indicates that the gel has polymerized. The whole process takes about 30 minutes (at 25℃).
3. Preparation of concentrated gel: first, the water on the upper layer of the polymerized separation gel is absorbed, and then the filter paper is used to blot out the residual liquid.
2. The separation glue solution (20ml, available for two plates) is prepared, and the mixture is shaken gently by hand. The mixture is carefully injected into the gap of the prepared glass plates, leaving enough space (~ 2.5cm) for the concentrated glue. The 0.5ml deionized water (or isopropanol) is gently added to the top layer to cover it, so as to prevent the inhibition of the condensation by oxygen in the air. It can be seen that there is an interface between the water and the gel when the water is first added, then it gradually disappears, and then the interface appears again soon, which indicates that the gel has polymerized. The whole process takes about 30 minutes (at 25℃).
3. Preparation of concentrated gel: first, the water on the upper layer of the polymerized separation gel is absorbed, and then the filter paper is used to blot out the residual liquid.
Preparation of concentrated gel solution. After mixing, it is injected into the top of the separating glue and inserted into the comb to avoid the appearance of bubbles.
4. During the concentrated gel polymerization, the protein sample was mixed with 4X sample buffer in equal volume, denaturated in 95℃ water bath or metal bath for 10min, and cooled to room temperature for use.
5. After the concentrated gel polymerization is complete, put the gel template into the electrophoresis tank and fix it. Add 1X electrophoresis buffer to both the upper and lower tanks.
Carefully pull out the comb, check for leaks and remove any bubbles
at the bottom of the gel between the glass panels.5. After the concentrated gel polymerization is complete, put the gel template into the electrophoresis tank and fix it. Add 1X electrophoresis buffer to both the upper and lower tanks.
3.1.2 Sampling and electrophoresis:
Steps：
1. Sample 20 ul/ well was added to each comb hole, and molecular weight marker 5ul was added to the first hole to react the protein size and monitor the electrophoresis process.
2. Electrophoresis: the voltage at the beginning is about 100V. After the dye is concentrated into a line and begins to enter the separation adhesive, the voltage is increased to about 160V, and the electrophoresis continues until the dye (bromophenol blue) reaches the bottom of the separation adhesive, and the power is disconnected.
3. Stripping: remove the rubber board, gently pry open the glass board from the bottom side, cut the concentrated glue with a matching plastic knife, and cut off a corner for marking, and soak in the pre-cooled electrotransfer buffer for balance.
2. Electrophoresis: the voltage at the beginning is about 100V. After the dye is concentrated into a line and begins to enter the separation adhesive, the voltage is increased to about 160V, and the electrophoresis continues until the dye (bromophenol blue) reaches the bottom of the separation adhesive, and the power is disconnected.
3. Stripping: remove the rubber board, gently pry open the glass board from the bottom side, cut the concentrated glue with a matching plastic knife, and cut off a corner for marking, and soak in the pre-cooled electrotransfer buffer for balance.
3.1.3 Transmembrane:
Cut one sheet of NC film of 4.5cm×8cm (cutting Angle marking direction) and six sheets of filter paper of 5cm×9cm, and soak them with the electrotransfer buffer precooled with ice in advance.
Steps：
Steps：
1. Connect the converter to the power supply, 105V for 1 hour.
3.1.4 Antibody incubation and detection:
1. The transfer membrane was cleaned twice with 1X TBS for 5min, and then the blocking solution was added for antibody sealing. The room temperature was 1 hour.
2. Primary antibody was incubated overnight at 4℃ (primary antibody was prepared with blocking solution).
3. On the second day, primary antibody was eluted; Secondary antibody was added and incubated in room temperature shaking table for 1h.
4. Eluting the secondary antibody, 5min each time, 5 times; Add chromogenic substrate for chemiluminescence chromogenic and image acquisition.
5. Exposure.
2. Primary antibody was incubated overnight at 4℃ (primary antibody was prepared with blocking solution).
3. On the second day, primary antibody was eluted; Secondary antibody was added and incubated in room temperature shaking table for 1h.
4. Eluting the secondary antibody, 5min each time, 5 times; Add chromogenic substrate for chemiluminescence chromogenic and image acquisition.
5. Exposure.
3.2 Dot Blot
A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or
paper substrate. Concentration of proteins in crude preparations (such as culture supernatant) can be estimated semi-quantitatively by using "Dot Blot" method if you have both purified protein and specific antibody against it.
Materials:
Materials:
1. TBS buffer:20 mM PH=7.5 Tris-HCl buffer.
2. TBST solution:0.05% Tween20 in TBS.
3. Soak 5% BSA in TBST, 0.5-1hr, RT, and place in a 10cm petri dish.
4. Incubate with primary Antibody (0.1-10 ug/ml for Antibody,1:1000 to 1:100000 Dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 30 min at RT.
Steps:
2. TBST solution:0.05% Tween20 in TBS.
3. Soak 5% BSA in TBST, 0.5-1hr, RT, and place in a 10cm petri dish.
4. Incubate with primary Antibody (0.1-10 ug/ml for Antibody,1:1000 to 1:100000 Dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 30 min at RT.
1. Take a nitrocellulose membrane and draw a grid on the acetate fiber membrane with a pencil.
2. The standard product will be diluted by multiple ratio, sample, negative control, positive control point in the middle of the grid on the membrane, and air dry.
3. Soak 5% BSA in TBST, 0.5-1hr, RT, and place in a 10cm petri dish.
4. 4. Incubate with primary Antibody (0.1-10 ug/ml for Antibody,1:1000 to 1:100000 Dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 30 min at RT.
5. Wash the nitrocellulose membrane twice with TBST buffer, and discard the solution as far as possible after each wash. Then wash with TBST buffer 3 times, 5 minutes each time.
6. After discarding the liquid, 30mL TBST buffer containing the second antibody (antibody dilution 1:2000) was added and incubated by shock at room temperature for 1 hour.
7. Fully wash the nitrocellulose membrane with TBST buffer twice, and discard the solution as far as possible after each wash. Then wash with TBST buffer 3 times, 5 minutes each time.
8. Remove the nitrocellulose membrane, slightly remove the excess solution on the membrane, and add an appropriate amount of fluorescent chromogen A and B (common with Western blot kit, generally 1/2 A film the size of 96-well plate plus 4mLA and B mixture, and liquid A: liquid B = 1:1).
9. Cover the nitrate cellulose film with cling film, as smooth as possible to avoid bubbles, and put it on the exposure clip, according to the brightness of the sample and the standard, initially determine the exposure time (under normal circumstances, if the sample brightness is visible to the naked eye and strong, then the first exposure time should be short, generally 3-5 seconds; If the brightness of the sample is not visible to the naked eye, the initial exposure time should be slightly longer, generally 1 minute), then take the appropriate size of the film covering the film, close the clip exposure.
10. After exposure, remove the film immersed in solution (ECL), and observed under red light until the sample point on the film no longer change, remove the film rinsed in water it (this step avoid solution mixed with fixing bath, reduce the fixing bath use efficiency), add the fixing bath, after the blue background transparent film, the film can be removed in running water flushing, wash and let dry.
2. The standard product will be diluted by multiple ratio, sample, negative control, positive control point in the middle of the grid on the membrane, and air dry.
3. Soak 5% BSA in TBST, 0.5-1hr, RT, and place in a 10cm petri dish.
4. 4. Incubate with primary Antibody (0.1-10 ug/ml for Antibody,1:1000 to 1:100000 Dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 30 min at RT.
5. Wash the nitrocellulose membrane twice with TBST buffer, and discard the solution as far as possible after each wash. Then wash with TBST buffer 3 times, 5 minutes each time.
6. After discarding the liquid, 30mL TBST buffer containing the second antibody (antibody dilution 1:2000) was added and incubated by shock at room temperature for 1 hour.
7. Fully wash the nitrocellulose membrane with TBST buffer twice, and discard the solution as far as possible after each wash. Then wash with TBST buffer 3 times, 5 minutes each time.
8. Remove the nitrocellulose membrane, slightly remove the excess solution on the membrane, and add an appropriate amount of fluorescent chromogen A and B (common with Western blot kit, generally 1/2 A film the size of 96-well plate plus 4mLA and B mixture, and liquid A: liquid B = 1:1).
9. Cover the nitrate cellulose film with cling film, as smooth as possible to avoid bubbles, and put it on the exposure clip, according to the brightness of the sample and the standard, initially determine the exposure time (under normal circumstances, if the sample brightness is visible to the naked eye and strong, then the first exposure time should be short, generally 3-5 seconds; If the brightness of the sample is not visible to the naked eye, the initial exposure time should be slightly longer, generally 1 minute), then take the appropriate size of the film covering the film, close the clip exposure.
10. After exposure, remove the film immersed in solution (ECL), and observed under red light until the sample point on the film no longer change, remove the film rinsed in water it (this step avoid solution mixed with fixing bath, reduce the fixing bath use efficiency), add the fixing bath, after the blue background transparent film, the film can be removed in running water flushing, wash and let dry.
3.3 SDS Page
3.3.1 Preparation of polyacrylamide gel:
Steps:
1. Installation of the plastic plate model: wash the glass plate, air dry, and spare. When making glue, choose the right glass plate and assemble the plastic plate model.
2. The separation glue solution (20ml, available for two plates) is prepared, and the mixture is shaken gently by hand. The mixture is carefully injected into the gap of the prepared glass plates, leaving enough space (~ 2.5cm) for the concentrated glue. The 0.5ml deionized water (or isopropanol) is gently added to the top layer to cover it, so as to prevent the inhibition of the condensation by oxygen in the air. It can be seen that there is an interface between the water and the gel when the water is first added, then it gradually disappears, and then the interface appears again soon, which indicates that the gel has polymerized. The whole process takes about 30 minutes (at 25℃).
3. Preparation of concentrated gel: first, the water on the upper layer of the polymerized separation gel is absorbed, and then the filter paper is used to blot out the residual liquid.
Preparation of concentrated gel solution. After mixing, it is injected into the top of the separating glue and inserted into the comb to avoid the appearance of bubbles.
4．During the concentrated gel polymerization, the protein sample was mixed with 4X sample buffer in equal volume, denaturated in 95℃ water bath or metal bath for 10min, and cooled to room temperature for use.
5．After the concentrated gel polymerization is complete, put the gel template into the electrophoresis tank and fix it. Add 1X electrophoresis buffer to both the upper and lower tanks.
6．Carefully pull out the comb, check for leaks and remove any bubbles at the bottom of the gel between the glass panels.
2. The separation glue solution (20ml, available for two plates) is prepared, and the mixture is shaken gently by hand. The mixture is carefully injected into the gap of the prepared glass plates, leaving enough space (~ 2.5cm) for the concentrated glue. The 0.5ml deionized water (or isopropanol) is gently added to the top layer to cover it, so as to prevent the inhibition of the condensation by oxygen in the air. It can be seen that there is an interface between the water and the gel when the water is first added, then it gradually disappears, and then the interface appears again soon, which indicates that the gel has polymerized. The whole process takes about 30 minutes (at 25℃).
3. Preparation of concentrated gel: first, the water on the upper layer of the polymerized separation gel is absorbed, and then the filter paper is used to blot out the residual liquid.
Preparation of concentrated gel solution. After mixing, it is injected into the top of the separating glue and inserted into the comb to avoid the appearance of bubbles.
4．During the concentrated gel polymerization, the protein sample was mixed with 4X sample buffer in equal volume, denaturated in 95℃ water bath or metal bath for 10min, and cooled to room temperature for use.
5．After the concentrated gel polymerization is complete, put the gel template into the electrophoresis tank and fix it. Add 1X electrophoresis buffer to both the upper and lower tanks.
6．Carefully pull out the comb, check for leaks and remove any bubbles at the bottom of the gel between the glass panels.
3.3.2 Sampling and electrophoresis:
Steps:
1. Sample 20 ul/ well was added to each comb hole, and molecular weight marker 5ul was added to the first hole to react the protein size and monitor the electrophoresis process.
2. Electrophoresis: the voltage at the beginning is about 100V. After the dye is concentrated into a line and begins to enter the separation adhesive, the voltage is increased to about 160V, and the electrophoresis continues until the dye (bromophenol blue) reaches the bottom of the separation adhesive, and the power is disconnected.
3. Cut glue. Cut the separating glue into a large plate with staining solution, stain for 1~2h, and then pour back the staining solution for repeated use. Then wash with UP first, and then decolorize with decolorizing solution for 4h (even overnight).
2. Electrophoresis: the voltage at the beginning is about 100V. After the dye is concentrated into a line and begins to enter the separation adhesive, the voltage is increased to about 160V, and the electrophoresis continues until the dye (bromophenol blue) reaches the bottom of the separation adhesive, and the power is disconnected.
3. Cut glue. Cut the separating glue into a large plate with staining solution, stain for 1~2h, and then pour back the staining solution for repeated use. Then wash with UP first, and then decolorize with decolorizing solution for 4h (even overnight).
3.4 Nickel column purification method
Purification method of protein with nickel column
Materials:
Materials:
The above reagents except 20% Ethanol should be filtered and sterilized by solvent filter, with a filter membrane of 0.45mm, and the filtration sequence was H2O→10mM PB(pH=7.4)→Binding buffer→Elution buffer.
3.4.1 Sample processing
E. coli: the day before purification, the bacteria (20 mL), which had been added lysozyme (1mg/mL), was taken out from -80℃, melted in water, and then add 200mL PMSF. The cells were broken by ultrasound, and the ultrasound lasted for 5s
and 5s, and the total ultrasonic time was 1min. After balancing, centrifuge at 10000g at 4℃ for 20min, take supernatant and pass through 0.45mm filter membrane. -80 ℃.
Pichia pastoris: Basically consistent with E.coli.
Crushing of precipitates: Centrifuge with 1ml bacterial solution, separate the supernatant from the bacteria, re-suspend the bacteria with 1mL PBS, take 200ul bacterial suspension +50ul 5*loading buffer, cook for 15-20min, centrifuge at 12000rpm for 10min (during the cooking process, put the tube on the float with the lid on. And select the induction cooker power is 300W, it is not easy to burst the tube).
Make 10mM PB, Binding buffer and Elution buffer, and save at 4℃ after filtering.
A box of sterilized EP tube, sampler, needle, absorbent paper, gloves, up water, corresponding column (His, desalting column, etc., note: column should be kept low temperature and moist) ready.
Steps:
Pichia pastoris: Basically consistent with E.coli.
Crushing of precipitates: Centrifuge with 1ml bacterial solution, separate the supernatant from the bacteria, re-suspend the bacteria with 1mL PBS, take 200ul bacterial suspension +50ul 5*loading buffer, cook for 15-20min, centrifuge at 12000rpm for 10min (during the cooking process, put the tube on the float with the lid on. And select the induction cooker power is 300W, it is not easy to burst the tube).
Make 10mM PB, Binding buffer and Elution buffer, and save at 4℃ after filtering.
A box of sterilized EP tube, sampler, needle, absorbent paper, gloves, up water, corresponding column (His, desalting column, etc., note: column should be kept low temperature and moist) ready.
Steps:
1. Since the N-terminal fusion of EGL7 expressed 6´His tags, HisTrap HP column was used to perform affinity chromatography on AKTA protein Purifier 10 (GE Healthcare).
2. Flush affinity column (column volume 1ml), system (system volume 10ml) and collection place (flow rate 1ml/min) with water.
3. Balance with the Binding Buffer.
4. The supernatant after filtration of the treated bacterial lysate is then pumped.
5. The affinity column was washed with Binding buffer to remove some of the mixed proteins with weak Binding ability.
6. After the protein peak A280 was stable, the target proteins were eluted by discontinuous gradient method: His column was eluted with the elution buffer containing 80 mM and 150 mM imidazole respectively to remove part of the untightly bound heteroproteins.
7. His column was eluted with elution buffer containing 500mM imidazole, and samples of the elution peak were collected on the ice for desalting (1 ml/tube).
2. Flush affinity column (column volume 1ml), system (system volume 10ml) and collection place (flow rate 1ml/min) with water.
3. Balance with the Binding Buffer.
4. The supernatant after filtration of the treated bacterial lysate is then pumped.
5. The affinity column was washed with Binding buffer to remove some of the mixed proteins with weak Binding ability.
6. After the protein peak A280 was stable, the target proteins were eluted by discontinuous gradient method: His column was eluted with the elution buffer containing 80 mM and 150 mM imidazole respectively to remove part of the untightly bound heteroproteins.
7. His column was eluted with elution buffer containing 500mM imidazole, and samples of the elution peak were collected on the ice for desalting (1 ml/tube).
3.4.2 His purification
Steps:
1. Turn on the computer, turn on the switch of the instrument (the light is all on means normal), and open the software UNICORN.
2. Select System Control, Manual, and Pump under Tools.
3. Put Pump A and Pump B in H2O and start flushing the pipeline system: system 1 → Pump→ Flow: 2mL/min (the flow rate can be increased to 10 mL/min), flush both pumps. Set Pump→Gradient: 50%-b, and Pump→ 0.2MPa(His column pressure does not exceed 0.3 MPa). Flush to the measured A280 and the salt trend is stable to be considered clean. The entire pipeline was filled with about 10 mL and took about 5min.
4. Flushing at the sampling ring: In the state of Flowpath→ Load, the injector should inject 1mL H2O (the actual injection ring used this time is 2mL, and no less than 2mL of water should be used for flushing), then change the state of Flowpath→ Inject, and Frac→Fraction900 should be set to 1mL/min to flush the collector.
5. Column washing: wash His column (column volume 1mL) and desalting column (column volume 5mL) with water, and try not to produce bubbles when installing the column. Set Pump→Gradient:0%B, Pump→Flow:1mL/min or 5mL/min (no more than the column pressure). If the measured A280 and the salt trend are stable, it is considered to be washed clean. Generally, two column volumes should be washed. Rinse the desalting column first, then rinse His column, so that the sample can be loaded directly after flushing.
6. Flush the system with the Binding buffer according to steps 3, 4 and 5.
7. Balanced His column: After pressing Pause, put Pump A into the Binding buffer and press continue, Pump→Flow:1mL/min to reach the measured A280 and the salt trend is stable.
8. Sample loading: After pressing Pause, put Pump A into the sample, Pump→Flow:1mL/min, and sample loading begins. At this time, A280 rises sharply and continues to end with the first sample (the penetration peak). Press Pause when the sample liquid level is close to the absorption point of pump A. After washing pump A clean with H2O, place it in the Binding buffer. Press continue to rinse until A280 and the salt trend is stable, and the impurities are washed away.
9. Elution: Determine that Pump A is in the Binding buffer, and Pump B is in the Elution buffer. After the stabilization of the previous step, zero correction is AlamsMon→AutoZeroUV, variable Flow rate Pump→Flow:1mL/min. Select the Elution concentration according to Pump→Gradient: X %B. Immediately select Frac→Fraction900, set it to 1mL/tube, focus on the 280 reading change (not the image itself), and select Execute to collect the sample once the A280 is trending up. After the collection, 500mM Elution buffer, namely Gradient:100%B, was used to wash away other impurities in the column, and His column was removed after A280 and the salt trend was stable.
10. Desalination: press Pause, after A pump is cleaned, put in 10mM PB, A280 and salt trend is stable after installing desalination column (G25), do not produce bubbles, A280 and salt trend is stable, set to Flowpath→load state, (the principle is: In the load state, the system liquid does not pass through the injection ring, and the liquid will remain in the injection ring. After being set to inject state, the injected liquid is equivalent to being washed away. Observe whether the load state or inject state and check the road diagram. The injector will absorb 1 mL of the sample collected in the previous step, remove the bubble and inject it into the injection ring. Set the state to Flowpath→ Inject, Frac→Fraction900 immediately and set it to 0.4ml /min (easy to lyophilize). If A280 has an upward trend, Select Execute to collect samples, Frac→Fraction900 set to 0mL/min when salt starts to rise, and stop collecting. Mark each tube with corresponding post-desalting sequence after elution and place on ice. If the image appears obvious bubble shape (280 straight line rises to high) after adding the sample, the desalting column can be removed before the next desalting, and then the desalting column can be installed after washing the pipeline.
Notes:
2. Select System Control, Manual, and Pump under Tools.
3. Put Pump A and Pump B in H2O and start flushing the pipeline system: system 1 → Pump→ Flow: 2mL/min (the flow rate can be increased to 10 mL/min), flush both pumps. Set Pump→Gradient: 50%-b, and Pump→ 0.2MPa(His column pressure does not exceed 0.3 MPa). Flush to the measured A280 and the salt trend is stable to be considered clean. The entire pipeline was filled with about 10 mL and took about 5min.
4. Flushing at the sampling ring: In the state of Flowpath→ Load, the injector should inject 1mL H2O (the actual injection ring used this time is 2mL, and no less than 2mL of water should be used for flushing), then change the state of Flowpath→ Inject, and Frac→Fraction900 should be set to 1mL/min to flush the collector.
5. Column washing: wash His column (column volume 1mL) and desalting column (column volume 5mL) with water, and try not to produce bubbles when installing the column. Set Pump→Gradient:0%B, Pump→Flow:1mL/min or 5mL/min (no more than the column pressure). If the measured A280 and the salt trend are stable, it is considered to be washed clean. Generally, two column volumes should be washed. Rinse the desalting column first, then rinse His column, so that the sample can be loaded directly after flushing.
6. Flush the system with the Binding buffer according to steps 3, 4 and 5.
7. Balanced His column: After pressing Pause, put Pump A into the Binding buffer and press continue, Pump→Flow:1mL/min to reach the measured A280 and the salt trend is stable.
8. Sample loading: After pressing Pause, put Pump A into the sample, Pump→Flow:1mL/min, and sample loading begins. At this time, A280 rises sharply and continues to end with the first sample (the penetration peak). Press Pause when the sample liquid level is close to the absorption point of pump A. After washing pump A clean with H2O, place it in the Binding buffer. Press continue to rinse until A280 and the salt trend is stable, and the impurities are washed away.
9. Elution: Determine that Pump A is in the Binding buffer, and Pump B is in the Elution buffer. After the stabilization of the previous step, zero correction is AlamsMon→AutoZeroUV, variable Flow rate Pump→Flow:1mL/min. Select the Elution concentration according to Pump→Gradient: X %B. Immediately select Frac→Fraction900, set it to 1mL/tube, focus on the 280 reading change (not the image itself), and select Execute to collect the sample once the A280 is trending up. After the collection, 500mM Elution buffer, namely Gradient:100%B, was used to wash away other impurities in the column, and His column was removed after A280 and the salt trend was stable.
10. Desalination: press Pause, after A pump is cleaned, put in 10mM PB, A280 and salt trend is stable after installing desalination column (G25), do not produce bubbles, A280 and salt trend is stable, set to Flowpath→load state, (the principle is: In the load state, the system liquid does not pass through the injection ring, and the liquid will remain in the injection ring. After being set to inject state, the injected liquid is equivalent to being washed away. Observe whether the load state or inject state and check the road diagram. The injector will absorb 1 mL of the sample collected in the previous step, remove the bubble and inject it into the injection ring. Set the state to Flowpath→ Inject, Frac→Fraction900 immediately and set it to 0.4ml /min (easy to lyophilize). If A280 has an upward trend, Select Execute to collect samples, Frac→Fraction900 set to 0mL/min when salt starts to rise, and stop collecting. Mark each tube with corresponding post-desalting sequence after elution and place on ice. If the image appears obvious bubble shape (280 straight line rises to high) after adding the sample, the desalting column can be removed before the next desalting, and then the desalting column can be installed after washing the pipeline.
1. All samples collected in step 8 were desalinated using the same method. The samples come out in almost the same position each time the salt is desalted.
2. Clean the column and line: wash the desalting column and His column with water until the salt is below 0.1. Inject 1mL of water into the sample ring in load state (Frac→Fraction900) and set it at 1mL/min. Clean the collector.
3. Finally, 20% Ethanol was used to clean the column and the whole pipeline, using the same method as above.
2. Clean the column and line: wash the desalting column and His column with water until the salt is below 0.1. Inject 1mL of water into the sample ring in load state (Frac→Fraction900) and set it at 1mL/min. Clean the collector.
3. Finally, 20% Ethanol was used to clean the column and the whole pipeline, using the same method as above.
Enzyme activity detection
4.1 An overview of enzyme activity determination
4.1.1 Expression of the amount and concentration of the enzyme
1.Expressed in molar quantities: x mol/L
2. Expressed by the activity of enzyme units:
2. Expressed by the activity of enzyme units:
①enzyme activity: under certain conditions, enzyme activity = the number of moles of substrate transformed/unit time
Unit of enzyme activity:
katal = 1mol / s
U=1μmol/min
1U = 16.67 nanocatals
U=1μmol/min
1U = 16.67 nanocatals
②Specific activity of enzyme: represents the purity of enzyme, the number of enzyme activity units per milligram of protein:
Specific activity of enzyme = enzyme activity U/protein mg
③the reaction rate of enzymes:
Enzyme reaction rate = substrate disappearance concentration/unit time
Enzyme activity = enzyme reaction rate × reaction volume
Specific activity of 100% pure enzyme/specific activity of impure protein sample
4.1.2 The principle of determining the catalytic activity of enzymes
1. What are the chemical changes that distinguish enzyme reactions?
Oxidation-reduction, Group Transfer, Elimination, Isomerization, Rearrangement, Condensation, Actualization
2. Consider whether there are chemical processes with similar mechanisms?
① Involving small molecular substrates or large molecular proteins or nucleic acids;
② Components that are easy to occur in solution or require membrane binding to carry out enzyme reaction;
③ To what extent the reverse reaction is carried out;
② Components that are easy to occur in solution or require membrane binding to carry out enzyme reaction;
③ To what extent the reverse reaction is carried out;
3. Essential principle: the ability to distinguish the physical and chemical properties of a given substrate and related products in a measurable way is designed.
4. Determination Centre: The central theme of enzyme activity detection is to ensure the determination of the initial rate. To reiterate this point, it is common and better practice to measure the initial rate of reactant reduction or product formation in a time with less than or equal to 5% product transformation. This approach can give a value closest to the initial reaction rate under the condition of initial substrate concentration.
4. Determination Centre: The central theme of enzyme activity detection is to ensure the determination of the initial rate. To reiterate this point, it is common and better practice to measure the initial rate of reactant reduction or product formation in a time with less than or equal to 5% product transformation. This approach can give a value closest to the initial reaction rate under the condition of initial substrate concentration.
4.1.3 Experimental materials
Enzyme samples, substrates, cofactors of enzymes
4.1.4 Experimental instruments
Water bath pot, pH meter, spectrophotometer, fluorescence photometer, calorimeter, HPLC
4.1.5 Experimental methods
Continuous methods
1. Spectrophotometry: Track the reaction process by measuring the change in the amount of light absorbed by the solution. For example: Because the common coenzymes NADH and NADPH absorb UV light in their reduced form, but not
in their oxidized form. Therefore, oxidoreductases using NADH as a substrate can be determined by reducing the UV absorbance at 340 nm wavelength with the consumption of coenzymes.
2. Colorimetry: If this light is in the visible area, the change of the measured color can actually be seen, and the reaction process can be tracked by measuring the color change of the measured solution. For example: MTT measurement.
3. Direct coupling assay: When the enzyme reaction does not cause a change in the absorbance of light, it is still possible to perform a spectrophotometric determination of the enzyme by using the coupling assay. Here, the product of one reaction is used as a substrate for another reaction that is easy to detect. For example, hexokinase is determined using glucose-6-phosphate dehydrogenase coupling.
4. Fluorescence method: Fluorescence is when a molecule emits light of one wavelength after absorbing different wavelengths of light. Fluorescence assays use the difference in the fluorescence of the substrate from the product to measure the enzyme reaction. These assays are usually more sensitive than spectrophotometric assays, but there are disadvantages, such as the possibility of suffering from interference caused by impurities, and the instability of many fluorescent compounds when exposed to light. Such as: An example of these assays is the use of nucleotide-coenzyme NADH and NADPH. Here, the reduced form is fluorescent and the oxidized form is non-fluorescent. Thus, the oxidation reaction can be carried out by measuring the decrease in fluorescence and the reduction reaction can be carried out by measuring the increase in fluorescence.
5. Calorimetry: Calorimetry is the measurement of the amount of heat released or absorbed by a chemical reaction. These assays are very versatile because many reactions involve some thermal change and use a microcalorimeter that does not require too many enzymes or substrates. These assays can be used to measure reactions that cannot be measured in any other way.
6. Chemiluminescence: Chemiluminescence is the emission of light by a chemical reaction. Some enzymatic reactions produce light, which can be measured to detect product formation. These types of assays can be very sensitive, as the generated light can be captured by photographic film for days or weeks, but may be difficult to quantify, as all light released by the reaction will not be detected. Detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method for detecting antibodies in Western blotting.
7. Light scattering method: Chemiluminescence is the emission of light by a chemical reaction. Some enzymatic reactions produce light, which can be measured to detect product formation. These types of assays can be very sensitive, as the generated light can be captured by photographic film for days or weeks, but may be difficult to quantify, as all light released by the reaction will not be detected. Detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method for detecting antibodies in Western blotting.
8. Microscale thermophoresis: Microscale thermophoresis (MST) measures the size, charge, and hydration entropy of the equilibrium time division molecule/substrate. The thermophoretic motion of the fluorescently labeled substrate varies significantly when modified by the enzyme. This enzyme activity can be measured in real time with high temporal resolution.] The material consumption of the all-optical MST method is very low, requiring only 5 μl sample volume and 10 nM enzyme concentration to measure the enzyme rate constants of activity and inhibition. MST allows the analyst to measure the modification of two different substrates at one time (multiplexing) If both substrates are labeled with different fluorophores. Matrix competition experiments can therefore be performed.
2. Colorimetry: If this light is in the visible area, the change of the measured color can actually be seen, and the reaction process can be tracked by measuring the color change of the measured solution. For example: MTT measurement.
3. Direct coupling assay: When the enzyme reaction does not cause a change in the absorbance of light, it is still possible to perform a spectrophotometric determination of the enzyme by using the coupling assay. Here, the product of one reaction is used as a substrate for another reaction that is easy to detect. For example, hexokinase is determined using glucose-6-phosphate dehydrogenase coupling.
4. Fluorescence method: Fluorescence is when a molecule emits light of one wavelength after absorbing different wavelengths of light. Fluorescence assays use the difference in the fluorescence of the substrate from the product to measure the enzyme reaction. These assays are usually more sensitive than spectrophotometric assays, but there are disadvantages, such as the possibility of suffering from interference caused by impurities, and the instability of many fluorescent compounds when exposed to light. Such as: An example of these assays is the use of nucleotide-coenzyme NADH and NADPH. Here, the reduced form is fluorescent and the oxidized form is non-fluorescent. Thus, the oxidation reaction can be carried out by measuring the decrease in fluorescence and the reduction reaction can be carried out by measuring the increase in fluorescence.
5. Calorimetry: Calorimetry is the measurement of the amount of heat released or absorbed by a chemical reaction. These assays are very versatile because many reactions involve some thermal change and use a microcalorimeter that does not require too many enzymes or substrates. These assays can be used to measure reactions that cannot be measured in any other way.
6. Chemiluminescence: Chemiluminescence is the emission of light by a chemical reaction. Some enzymatic reactions produce light, which can be measured to detect product formation. These types of assays can be very sensitive, as the generated light can be captured by photographic film for days or weeks, but may be difficult to quantify, as all light released by the reaction will not be detected. Detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method for detecting antibodies in Western blotting.
7. Light scattering method: Chemiluminescence is the emission of light by a chemical reaction. Some enzymatic reactions produce light, which can be measured to detect product formation. These types of assays can be very sensitive, as the generated light can be captured by photographic film for days or weeks, but may be difficult to quantify, as all light released by the reaction will not be detected. Detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method for detecting antibodies in Western blotting.
8. Microscale thermophoresis: Microscale thermophoresis (MST) measures the size, charge, and hydration entropy of the equilibrium time division molecule/substrate. The thermophoretic motion of the fluorescently labeled substrate varies significantly when modified by the enzyme. This enzyme activity can be measured in real time with high temporal resolution.] The material consumption of the all-optical MST method is very low, requiring only 5 μl sample volume and 10 nM enzyme concentration to measure the enzyme rate constants of activity and inhibition. MST allows the analyst to measure the modification of two different substrates at one time (multiplexing) If both substrates are labeled with different fluorophores. Matrix competition experiments can therefore be performed.
4.1.6 Note:
Factors that may affect the determination:
1. Salt concentration: Most enzymes cannot tolerate extremely high salt concentrations. Ions interfere with proteins with weak ion bonds. Typical enzymes are active at salt concentrations of 1-500 mM. For example, salinity, algae and
bacteria.
2. Effect of temperature: All enzymes function within a temperature range specific to the organism. Increased temperature usually leads to an increase in the rate of reaction. Higher temperatures lead to a sharp decrease in the rate of reaction. This is due to the decomposition of weak ions and hydrogen bonds Denaturation (alteration) of the protein structure that stabilizes the three-dimensional structure of the check point of enzyme activity. The "optimal" temperature for human enzymes is usually between 35 and 40 ° C. The average temperature in humans is 37 ℃. Human enzymes begin to denature rapidly at temperatures above 40 ℃. From thermophilic enzymes, archaea found in hot springs are stable at up to 100 ℃. The idea of an "optimal" rate for enzyme reaction is unreasonable because the rate observed at any temperature is the product of two rates, the rate of reaction and the rate of denaturation.
3. Effect of pH: Most enzymes are sensitive to pH and have a specific range of activities. All have an optimal pH value. pH can prevent enzyme activity by deforming (changing) the three-dimensional shape of the enzyme by breaking the ionic and hydrogen bonds. Most enzymes have pH values between 6 and 8; however, pepsin in the stomach works best at pH 2 and trypsin works best at pH 8.
4. Substrate saturation: Increasing the substrate concentration can increase the reaction rate (enzyme activity). However, the enzyme saturation limits the reaction rate. When most of the time the activity check point of all molecules is occupied, the enzyme will be saturated. At the saturation point, no matter how many additional substrates are added, the reaction will not accelerate. The reaction rate graph will tend to be stable.
5. The crowding effect of macromolecules. The crowding degree of a large number of macromolecules in the solution will change the rate and equilibrium constant of the enzyme reaction.
2. Effect of temperature: All enzymes function within a temperature range specific to the organism. Increased temperature usually leads to an increase in the rate of reaction. Higher temperatures lead to a sharp decrease in the rate of reaction. This is due to the decomposition of weak ions and hydrogen bonds Denaturation (alteration) of the protein structure that stabilizes the three-dimensional structure of the check point of enzyme activity. The "optimal" temperature for human enzymes is usually between 35 and 40 ° C. The average temperature in humans is 37 ℃. Human enzymes begin to denature rapidly at temperatures above 40 ℃. From thermophilic enzymes, archaea found in hot springs are stable at up to 100 ℃. The idea of an "optimal" rate for enzyme reaction is unreasonable because the rate observed at any temperature is the product of two rates, the rate of reaction and the rate of denaturation.
3. Effect of pH: Most enzymes are sensitive to pH and have a specific range of activities. All have an optimal pH value. pH can prevent enzyme activity by deforming (changing) the three-dimensional shape of the enzyme by breaking the ionic and hydrogen bonds. Most enzymes have pH values between 6 and 8; however, pepsin in the stomach works best at pH 2 and trypsin works best at pH 8.
4. Substrate saturation: Increasing the substrate concentration can increase the reaction rate (enzyme activity). However, the enzyme saturation limits the reaction rate. When most of the time the activity check point of all molecules is occupied, the enzyme will be saturated. At the saturation point, no matter how many additional substrates are added, the reaction will not accelerate. The reaction rate graph will tend to be stable.
5. The crowding effect of macromolecules. The crowding degree of a large number of macromolecules in the solution will change the rate and equilibrium constant of the enzyme reaction.
4.2 Protein concentration
We used ammonium sulfate precipitation for protein concentration.
Materials:
Materials:
① Ammonium Sulfate(saturated solution or solids)
② Fermentation Supernatant
③ Protein Resuspension Buffer: 20 mM HEPES, 10% glycerol, 500 mM NaCl, pH 7.5 (the isoelectric point of each protein is different, so the H needs to be set at the non-isoelectric point)
② Fermentation Supernatant
③ Protein Resuspension Buffer: 20 mM HEPES, 10% glycerol, 500 mM NaCl, pH 7.5 (the isoelectric point of each protein is different, so the H needs to be set at the non-isoelectric point)
Pre-experiment:
Goal: Estimating the final concentration of ammonium sulfate required for protein precipitation.
Steps:
Goal: Estimating the final concentration of ammonium sulfate required for protein precipitation.
Steps:
① Mix saturated ammonium sulfate solution with fermentation supernatant to final concentration of ammonium sulfate:10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%.
② Stand at 4 ℃ for 4~6h to precipitate the target protein.
Attention: standing for more than 6h would result in precipitating other proteins.
③ 4 ℃, 12000rpm for 20 min in refrigerated centrifuge.
④ Discard supernatant and resuspend the precipitation using protein resuspension buffer.
⑤ If the target protein is an enzyme, its enzyme activity should be detected; If not, western blot is necessary to compare the amount of it and choose the best final concentration of ammonium sulfate.
② Stand at 4 ℃ for 4~6h to precipitate the target protein.
Attention: standing for more than 6h would result in precipitating other proteins.
③ 4 ℃, 12000rpm for 20 min in refrigerated centrifuge.
④ Discard supernatant and resuspend the precipitation using protein resuspension buffer.
⑤ If the target protein is an enzyme, its enzyme activity should be detected; If not, western blot is necessary to compare the amount of it and choose the best final concentration of ammonium sulfate.
Formal experiment:
Goal: precipitate large amount of fermentation supernatant.
Steps:
Goal: precipitate large amount of fermentation supernatant.
Steps:
① Place the supernatant in a beaker filled with ice-water mixture and place it on a magnetic stirrer for continuous stirring.
② The ammonium sulfate solids required for the optimal final concentration of ammonium sulfate were uniformly added to the sample within 5-10min, and then stirred continuously for 20-30min.
Attention: the stirring should be mild and stable, and if a large amount of foam appears or the liquid heats up, the protein may have denatured.
③ Placed at 4 ℃ for 4~6h.
③ Centrifuge at 4 ℃ for 20 min.
④ Discard the supernatant and add appropriate amount of protein resuspend buffer to resuspend the pellet.
⑤ Add PMSF and save at -80 ℃. Avoid repeated freezing and thawing.
② The ammonium sulfate solids required for the optimal final concentration of ammonium sulfate were uniformly added to the sample within 5-10min, and then stirred continuously for 20-30min.
Attention: the stirring should be mild and stable, and if a large amount of foam appears or the liquid heats up, the protein may have denatured.
③ Placed at 4 ℃ for 4~6h.
③ Centrifuge at 4 ℃ for 20 min.
④ Discard the supernatant and add appropriate amount of protein resuspend buffer to resuspend the pellet.
⑤ Add PMSF and save at -80 ℃. Avoid repeated freezing and thawing.
4.3 Method for determination of xylanase activity
Preparation:
1. Configure the DNS reagent one week in advance.
2. Prepare 1% xylan solution, 50mmol/ L NaAC-HAC buffer, 50mmol/ L pH=5, xylanase solution and 1mg/ mL xylose solution:
2. Prepare 1% xylan solution, 50mmol/ L NaAC-HAC buffer, 50mmol/ L pH=5, xylanase solution and 1mg/ mL xylose solution:
①Preparation of NaAC-HAC buffer with 50mmol/ L pH=5:
4.1g NaAC and 1.6mL HAC with ddH2O to 1L.
②Preparation of 1% xylan solution:
1g xylan to 100ml.
③Preparation of xylanase solution:
1g xylanase powder was fully ground and dissolved in 40m, and NaAC-HAC buffer solution. A magnetic agitator was used to stir it for 30min. After centrifugation for 30s-1min at a low speed, the supernatant was taken, which was the crude
enzyme solution.
④ Preparation of 1mg/ml xylose solution.
3. Prepare a 50℃ water bath in advance.
4. Prepare a boiling water bath during the experiment:
5. Formal experiment:
4. Prepare a boiling water bath during the experiment:
5. Formal experiment:
(1)The control group (blank group) (50mlBD tube was used for the experiment):
①Add 1ml NaAC-HAC buffer.
②Add 0.5ml xylanase solution (OD520).
③Add 2mlDNS reagent and shake the test tube slightly.
④Add 0.5ml 1% xylan solution.
⑤Heat preservation at 50℃ for 5min.
⑥ Cool with tap water to room temperature.
⑦Drop to 96 well plate to determine OD520 absorption value EA.
②Add 0.5ml xylanase solution (OD520).
③Add 2mlDNS reagent and shake the test tube slightly.
④Add 0.5ml 1% xylan solution.
⑤Heat preservation at 50℃ for 5min.
⑥ Cool with tap water to room temperature.
⑦Drop to 96 well plate to determine OD520 absorption value EA.
(2) The experimental group:
①Add 1ml NaAC-HAC buffer.
②Add 0.5ml xylanase solution (OD520).
③Add 0.5ml 1% xylan solution.
④Heat preservation at 50℃ for 5min.
⑤Add 2ml DNS reagent and shake the tube slightly.
⑥ Cool with tap water to room temperature.
⑦Add to the 96-well plate to determine the absorption value EB of OD520.
(Theoretically heavier color, less xylan, more xylose, and higher ABSORPTION EB).
②Add 0.5ml xylanase solution (OD520).
③Add 0.5ml 1% xylan solution.
④Heat preservation at 50℃ for 5min.
⑤Add 2ml DNS reagent and shake the tube slightly.
⑥ Cool with tap water to room temperature.
⑦Add to the 96-well plate to determine the absorption value EB of OD520.
(Theoretically heavier color, less xylan, more xylose, and higher ABSORPTION EB).
(3) Determination of xylose standard line:
①Prepare 1mg/ml xylose solution.
②take 0,0.1, 0.2, 0.3, 0.4, 0.5, 0.6ml and add 1ml with ddH2O.
③Add 1mlDNS reagent respectively.
④Boiling water bath for 5min.
⑤Cool with tap water to room temperature.
⑥Drop to 96 well plate to determine OD520 absorption value.
②take 0,0.1, 0.2, 0.3, 0.4, 0.5, 0.6ml and add 1ml with ddH2O.
③Add 1mlDNS reagent respectively.
④Boiling water bath for 5min.
⑤Cool with tap water to room temperature.
⑥Drop to 96 well plate to determine OD520 absorption value.
(4) Calculate enzyme activity:
Xc = [(EB - EA) * K + D0] x 1000 / (M (t)
Among them:
EA group
EB group
K: Slope of xylose standard curve
D0: Intercept of xylose standard curve
M: Molar mass of xylose, g/mol
T:Reaction time
EA group
EB group
K: Slope of xylose standard curve
D0: Intercept of xylose standard curve
M: Molar mass of xylose, g/mol
T:Reaction time
4.4 Alkali titration for determination of lipase activity
We apply alkali titration method to determine the enzyme activity of lipase refer to GB/T23535-2009 method and the principle is that lipase can decompose oils into fatty acids which can react with alkalis. According to the standard, a lipase enzyme activity
unit(U) is the amount of lipase producing 1 umol fatty acid per minute under certain reaction conditions which can be determined by the volume consumed by NaOH.
Steps:
Steps:
1. Configure 4% polyvinyl alcohol (PVA) solution, PH=7.5 Phosphate buffer, 95% ethanol solution and 0.1 mol/L NaOH standard solution.
2. Mix olive oil and 4% polyvinyl alcohol (PVA) solution in a volume ratio of 1:3 and stir the mixture with a high-speed homogenizer to obtain a milky white emulsion which is the substrate for lipase hydrolysis.
3. Take 1ml fermentation supernatant or 0.1ml diluted lipase solution, add 4ml substrate solution,5ml PH=7.5 phosphate buffer and mix.
4. Put the mixture into a preheated 40 ℃ water bath, place it for 15minutes.
5. Terminate the reaction with 15 ml 95% ethanol solution.
6. Add 2 drops phenolphthalein indicator, titrate with 0.1mol/L NaOH standard solution until the liquid is slightly red and does not fade within 30s, then record the volume 0.1 mol/L NaOH standard solution consumed.
7. Calculate lipase activity on the basis of the volume 0.1 mol/L NaOH standard solution consumed.
2. Mix olive oil and 4% polyvinyl alcohol (PVA) solution in a volume ratio of 1:3 and stir the mixture with a high-speed homogenizer to obtain a milky white emulsion which is the substrate for lipase hydrolysis.
3. Take 1ml fermentation supernatant or 0.1ml diluted lipase solution, add 4ml substrate solution,5ml PH=7.5 phosphate buffer and mix.
4. Put the mixture into a preheated 40 ℃ water bath, place it for 15minutes.
5. Terminate the reaction with 15 ml 95% ethanol solution.
6. Add 2 drops phenolphthalein indicator, titrate with 0.1mol/L NaOH standard solution until the liquid is slightly red and does not fade within 30s, then record the volume 0.1 mol/L NaOH standard solution consumed.
7. Calculate lipase activity on the basis of the volume 0.1 mol/L NaOH standard solution consumed.
4.5 Method for determination of laccase activity
We apply ABTS to detect the enzyme activity of laccase and the principle is that laccase can decompose ABTS into ABTS free radicals whose absorption coefficient at 420 nm is much greater than that of the substrate ABTS. As the concentration of ABTS free
radicals increases, the absorbance value becomes larger. According to the standard, a laccase enzyme activity unit(U) is the change of the conversion of 1umol ABTS to ABTS free radicals which can be detected by the change in absorbance
value.
Steps:
Steps:
1. Configure 0.5mmol/L ABTS solution and PH=4.5 Hac-NaAc buffer.
2. Take 1ml fermentation supernatant or 0.1ml diluted laccase solution, add 2.5ml Hac-NaAc buffer and mix.
3. Put the mixture into a preheated 40 ℃ water bath, add 0.4ml ABTS solution and place it for 30s.
4. Measure the absorbance at 420 nm every 15 seconds for 6-7 times and draw a curve to calculate the slope.
5. Calculate laccase activity on the basis of the slope.
2. Take 1ml fermentation supernatant or 0.1ml diluted laccase solution, add 2.5ml Hac-NaAc buffer and mix.
3. Put the mixture into a preheated 40 ℃ water bath, add 0.4ml ABTS solution and place it for 30s.
4. Measure the absorbance at 420 nm every 15 seconds for 6-7 times and draw a curve to calculate the slope.
5. Calculate laccase activity on the basis of the slope.
4.6 Method for determination of cellulase activity
The enzyme activity of cellulase was determined by the method of DNS determination of reducing sugar (Miller G.L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 1959;31:426–428. doi: 10.1021/ac60147a030), and the
enzymatic reaction was determined according to Bernardi et al. (https://pubmed.ncbi.nlm.nih.gov/29715559/) The reaction system consisted of 1% (w/v) cm-cellulose in 50mM sodium acetate buffer (pH=5.0) and was held at 55℃ for 10min.
Add equal volume of DNS reagent to stop enzyme action. The mixture was boiled for 5min and cooled, and the absorbance was measured at 540 nm. One unit of endoglucanase activity is defined as the enzyme amount of releasing 1mol of reducing
sugar per minute. Each experiment was repeated three times. The protein concentration was determined by the Greenberg method (18. David B.Y. The colorimetric determination of the serum proteins. J. Biol. Chem. 1929;82:545–550).
Deinking section
Due to time limits, we first used commercial cellulase, xylanase, laccase and lipase to conduct in-situ deinking experiments, first explored the method of in-situ deinking, and promoted the hardware design of the project.
In order to explore the effect of enzymes on paper deinking, a piece of A4 paper was divided into small cells of the same size（see in Fig.1）, each containing the same number of points. Control experiments were designed. The bevel device is used to process
the paper after soaking, and the sticky cylinder falls free under the flat paper to take the ink. It is known that when the cylinder rolls around, it just completely covers 6×7 small cells, so 42 small cells can be obtained by each
operation of a piece of paper, and the number of residual ink spots in it is counted.
You can see statistics of this method at model1.
Figure.1 A4 paper division
Purification
To obtain the purified protein through the purification column when it has been determined by protein detection and enzyme activity detection that the bacteria can secrete sufficient amount of the target protein．
Materials:
Materials:
1. Protein Purification Instrument
2. Protein concentration column
3. ddH2O
4. buffer A and buffer C
5. Desalination column
6. 20% Ethanol
7. 50mM HEPES（PH=8）
2. Protein concentration column
3. ddH2O
4. buffer A and buffer C
5. Desalination column
6. 20% Ethanol
7. 50mM HEPES（PH=8）
Steps:
1. Use Buffer A to flush and balance the column until the baseline level.
2. Sample the crude body fluid of enzyme after filtration (0.45μm).
3. Buffer A was used to wash the hybrid protein that was not bound to Ni2+ column. At this time, the hybrid protein peak would appear, and the sample was taken by 10mL test tube.
4. After reaching the baseline level and stabilizing at 10mL, Buffer C was used to elute the protein bound to the Ni2+ column, and 2mL tube was used to sample when the peak was out.
5. After elution, flush the line with suction and filtration water and stabilize 10-20ml.
6. Flush the line with 20% ethanol and stabilize 10-20ml.
7. The desalting column was washed once with 20% ethanol.
8. Filtrate and wash three times.
9. Wash 50 mM TRIS-SO4 (PH: 8.0) three times.
10. Add 1mL purified protein and drain.
11. Add 1.5mL 50 mM TRIS-SO4 (PH: 8.0) to sample 1.5mL. The desalted protein is obtained at 20 ℃.
12. Wash and desalinate the column for repeated use.
13. Wash 3 times with 50 mM TRIS-SO4 (PH: 8.0).
14. Filtrate and wash three times.
15. The desalting column was washed once with 20% ethanol.
16. The desalinated column was preserved with 20% ethanol.
2. Sample the crude body fluid of enzyme after filtration (0.45μm).
3. Buffer A was used to wash the hybrid protein that was not bound to Ni2+ column. At this time, the hybrid protein peak would appear, and the sample was taken by 10mL test tube.
4. After reaching the baseline level and stabilizing at 10mL, Buffer C was used to elute the protein bound to the Ni2+ column, and 2mL tube was used to sample when the peak was out.
5. After elution, flush the line with suction and filtration water and stabilize 10-20ml.
6. Flush the line with 20% ethanol and stabilize 10-20ml.
7. The desalting column was washed once with 20% ethanol.
8. Filtrate and wash three times.
9. Wash 50 mM TRIS-SO4 (PH: 8.0) three times.
10. Add 1mL purified protein and drain.
11. Add 1.5mL 50 mM TRIS-SO4 (PH: 8.0) to sample 1.5mL. The desalted protein is obtained at 20 ℃.
12. Wash and desalinate the column for repeated use.
13. Wash 3 times with 50 mM TRIS-SO4 (PH: 8.0).
14. Filtrate and wash three times.
15. The desalting column was washed once with 20% ethanol.
16. The desalinated column was preserved with 20% ethanol.
Notes:
1. After the purified protein is obtained, the imidazole in the protein solution needs to be removed, because the imidazole will make the enzyme easy to precipitate, so the imidazole is washed with a desalination column and replaced
with Buffer, so that the purified enzyme can be preserved for a long time.
2. Use SDS-page to determine whether the acquired protein is the target protein.
2. Use SDS-page to determine whether the acquired protein is the target protein.
Myc label protein was detected by immunoprecipitation method
1. Re-suspend anti-Myc affinity gel to homogenize, transfer 20uL mixture (containing about 10uL gel) to centrifugal tube, add 10 times gel volume (about 100uL) PBS, 5000rpm x 30sec, discard supernatant, repeat this step to clean gel three times.
2. Add 50-200ul eukaryotic cell lysate containing target protein, and incubate at room temperature for 2hr or 4C overnight.
3. Centrifugal separation gel transfer the supernatant to a new EP tube for use, and the supernatant can be used to detect whether there is residual Myc-Tag protein.
4. Wash the gel
Add 10 times the gel PBS, and clean the gel three times by centrifugal method. The gel was washed by pre-cooling 5 times gel volume with pH 5.0 acidic prewash to remove non-specific binding proteins. Centrifuge supernatant.
5. Add 2x loading buffer and centrifuge. Boil for 5min, cool to room temperature, take supernatant for SDS-PAGE or WB detection.
2. Add 50-200ul eukaryotic cell lysate containing target protein, and incubate at room temperature for 2hr or 4C overnight.
3. Centrifugal separation gel transfer the supernatant to a new EP tube for use, and the supernatant can be used to detect whether there is residual Myc-Tag protein.
4. Wash the gel
Add 10 times the gel PBS, and clean the gel three times by centrifugal method. The gel was washed by pre-cooling 5 times gel volume with pH 5.0 acidic prewash to remove non-specific binding proteins. Centrifuge supernatant.
5. Add 2x loading buffer and centrifuge. Boil for 5min, cool to room temperature, take supernatant for SDS-PAGE or WB detection.
Co-Immunoprecipitation
Co-Immunoprecipitation is a classical method used to study protein interaction based on the specific interaction between antibody and antigen. It is an effective method to determine the rational interaction between
two proteins in intact cells.
The idea is that when cells are cleaved under non-denatured conditions, many of the protein-protein interactions present in intact cells are retained. If X is immunoprecipitated with antibodies to protein X, the protein Y bound to X in
vivo can also precipitate.
Materials:
1. Pre-cooling PBS
2. RIPA Buffer
3. Cell scraper (wrapped with plastic wrap, buried under ice)
4. Centrifuge Steps:
2. RIPA Buffer
3. Cell scraper (wrapped with plastic wrap, buried under ice)
4. Centrifuge Steps:
Steps:
1. Cells were washed twice with pre-cooled PBS, and PBS was sucked dry for the last time;
2. Add pre-cooled RIPA Buffer(1ml/107 cells, 10cm dish or 150cm2 culture bottle, 0.5ml/5×106 cells, 6cm dish and 75cm2 culture bottle)
3. Scrape the cells from the culture dish or flask with a pre-cooled cell scraper, transfer the suspension to 1.5eP tube, 4℃, slowly shake for 15min (EP tube inserted on ice, placed on a horizontal shaker)
4. Centrifuge at 14000g at 4℃ for 15min, immediately transfer the supernatant to a new centrifuge tuber>5. Prepare Protein A agarose, wash the beads twice with PBS, then prepare them with PBS to make 50% concentration. It is recommended to remove the tip part to avoid damaging the agarose beads in the operation involving agarose beads
6. Add 100μ L Protein A agarose beads (50%) to each 1ml of total Protein, shake at 4℃ for 10min (EP tube on ice, horizontal shaker) to remove non-specific impurities and reduce background
7. Centrifuge at 14000g at 4℃ for 15min, transfer the supernatant to A new centrifuge tube and remove Protein A beads
8. (Bradford method) Make protein standard curve, measure protein concentration, dilute total protein at least 1:10 times before measurement, to reduce the influence of detergent in cell lysis solution (quantitative, after packaging, can be stored at -20℃ for one month)
9. Dilute the total protein with PBS to about 1 μg/μ L to reduce the concentration of detergent in the lysate. If the protein of interest is low in the cell, the total protein concentration should be slightly higher (e.g. 10 μg/μ L).
10. Add a certain volume of rabbit antibody to 500μ L of total protein. The dilution ratio of antibody varies with the amount of protein of interest in different cell lines
11. Slowly shake the antigen/antibody mixture at 4℃ overnight or at room temperature for 2h. Analysis of kinase or phosphatase activity suggests incubation at room temperature for 2h
12. Add 100μ L Protein A agarose beads to capture the antigenic antibody complex, shake the antigenic antibody mixture at 4℃ overnight or at room temperature for 1h, if the antibody is mouse or chicken, add 2 μ L "transition antibody" (Rabbit anti-mouse IgG, Rabbit anti-chicken IgG)
13. Centrifuge at 14000rpm for 5s, collect agarose bead - antigen antibody complex, remove supernatant, wash 3 times with pre-cooled RIPA Buffer, 800μ L/time, RIPA Buffer sometimes will destroy the internal binding of agarose bead - antigen antibody complex, PBS can be used by>14. Suspend agarose bead - antigen - antibody complex with 60μ L 2× sample loading buffer and mix lightly. The amount of buffer depends on the amount of sample loading (60μ L is enough for three doses)
15. Cook the samples for 5min to free antigens, antibodies, beads, centrifuge, and electrophoresis the supernatant to collect the remaining agarose beads. The supernatant can also be temporarily frozen at -20℃ and left for later electrophoresis, which should be boiled again for 5min before electrophoresis to denature
2. Add pre-cooled RIPA Buffer(1ml/107 cells, 10cm dish or 150cm2 culture bottle, 0.5ml/5×106 cells, 6cm dish and 75cm2 culture bottle)
3. Scrape the cells from the culture dish or flask with a pre-cooled cell scraper, transfer the suspension to 1.5eP tube, 4℃, slowly shake for 15min (EP tube inserted on ice, placed on a horizontal shaker)
4. Centrifuge at 14000g at 4℃ for 15min, immediately transfer the supernatant to a new centrifuge tuber>5. Prepare Protein A agarose, wash the beads twice with PBS, then prepare them with PBS to make 50% concentration. It is recommended to remove the tip part to avoid damaging the agarose beads in the operation involving agarose beads
6. Add 100μ L Protein A agarose beads (50%) to each 1ml of total Protein, shake at 4℃ for 10min (EP tube on ice, horizontal shaker) to remove non-specific impurities and reduce background
7. Centrifuge at 14000g at 4℃ for 15min, transfer the supernatant to A new centrifuge tube and remove Protein A beads
8. (Bradford method) Make protein standard curve, measure protein concentration, dilute total protein at least 1:10 times before measurement, to reduce the influence of detergent in cell lysis solution (quantitative, after packaging, can be stored at -20℃ for one month)
9. Dilute the total protein with PBS to about 1 μg/μ L to reduce the concentration of detergent in the lysate. If the protein of interest is low in the cell, the total protein concentration should be slightly higher (e.g. 10 μg/μ L).
10. Add a certain volume of rabbit antibody to 500μ L of total protein. The dilution ratio of antibody varies with the amount of protein of interest in different cell lines
11. Slowly shake the antigen/antibody mixture at 4℃ overnight or at room temperature for 2h. Analysis of kinase or phosphatase activity suggests incubation at room temperature for 2h
12. Add 100μ L Protein A agarose beads to capture the antigenic antibody complex, shake the antigenic antibody mixture at 4℃ overnight or at room temperature for 1h, if the antibody is mouse or chicken, add 2 μ L "transition antibody" (Rabbit anti-mouse IgG, Rabbit anti-chicken IgG)
13. Centrifuge at 14000rpm for 5s, collect agarose bead - antigen antibody complex, remove supernatant, wash 3 times with pre-cooled RIPA Buffer, 800μ L/time, RIPA Buffer sometimes will destroy the internal binding of agarose bead - antigen antibody complex, PBS can be used by>14. Suspend agarose bead - antigen - antibody complex with 60μ L 2× sample loading buffer and mix lightly. The amount of buffer depends on the amount of sample loading (60μ L is enough for three doses)
15. Cook the samples for 5min to free antigens, antibodies, beads, centrifuge, and electrophoresis the supernatant to collect the remaining agarose beads. The supernatant can also be temporarily frozen at -20℃ and left for later electrophoresis, which should be boiled again for 5min before electrophoresis to denature
If this experiment is successful, we can not only avoid human influence to verify the interaction between scaffoldin and enzymes but also acquire interaction protein complexes in their natural state.