1 Medium
- LB (Luria-Bertani) medium
| Component | Amount |
|---|---|
| NaCl | 10 g/L |
| Tryptone | 10 g/L |
| Yeast extract | 5 g/L |
| Agar | 20 g/L |
- GYT medium
| Component | Amount |
|---|---|
| Glycerin | 0.1 mL/mL |
| Yeast extract | 0.00125 g/mL |
| Tryptone | 0.0025 g/mL |
- Separation gel (15%)
| Component | Amount |
|---|---|
| Ultra pure water | 0.5 mL/gel |
| 30% Acr-Bis (29:1) | 2.5 mL/gel |
| 1M Tris, pH8.8 | 1.9 mL/gel |
| 10% SDS | 0.05 mL/gel |
| 10% Gel polymerization catalyst | 0.05 mL/gel |
- Spacer gel
| Component | Amount |
|---|---|
| Ultra pure water | 1.4 mL/gel |
| 30% Acr-Bis (29:1) | 0.33 mL/gel |
| 1M Tris, pH8.8 | 0.25 mL/gel |
| 10% SDS | 0.02 mL/gel |
| 10% Gel polymerization catalyst | 0.02 mL/gel |
| TEMED | 0.002 mL/gel |
- Electrophoretic buffer solution
| Component | Amount |
|---|---|
| Tris | 30 g/L |
| Glycine | 144 g/L |
| SDS | 10 g/L |
- 1×TBST
| Component | Amount |
|---|---|
| 10×TBST | 100 mL/L |
| Tween | 5 mL |
- 5% BSA
| Component | Amount |
|---|---|
| BSA | 50 g/L |
| TBST | 1000 mL/L |
2 Electro transformation
a. Divide Nissle E.coli suspension (OD600 was 0.6-0.8) into 2 tubes.
b. Cool them with ice for 15 minutes.Di
c. Cool ultra pure water, 10% glycerin and EP tube.
d. Centrifuge at 3000 rpm for 10 minutes, then discard supernatant.
e. Re-suspend with ultra pure water.
f. Centrifuge at 4000 rpm for 10 minutes, then discard supernatant.
g. Re-suspend with 10% glycerin.
h. Centrifuge at 4000 rpm for 10 minutes, then discard supernatant.
i. Add 50 μL GYT in each tube, and then transfer them into cooled EP tube.
j. Add 20 μL ultra pure water to plasmid powder, then take 3 μL and add into the E.coli prepared.
k. Cool it with ice for 5 minutes, then add to electroporation instrument to carry out the electrotransformation.
l. Add 1 mL LB medium to re-suspend, then transfer it into EP tube.
3 SDS-PAGE
a. Use the BCA kit to check the concentration of the protein.
b. Add 6 μL RIPA and 6 μL 5× buffer to 18 μL E.coli suspension, then heat the sample at 100 ℃ for 10 minutes.
c. Add 10 μL sample to each hole on the gel.
d. Perform electrophoresis (80 V for 30 minutes, then 120 V for about 70 minutes).
e. Put the gel into the solution contained Coomassie Brilliant Blue and shake for several hours.
f. Discard the Coomassie Brilliant Blue solution. Add destainer and stand overnight.
4 Western Blot
a. Perform SDS-PAGE.
b. Wash the nitrocellulose membrane with methyl alcohol for 1-2 min at room temperature.
c. Transfer protein onto the membrane by electric under 120 V, 300 mA (cool with ice).
d. Wash the membrane with TBST, then stain it.
e. Wash the membrane with TBST.
f. Block it with 5% BSA for an hour.
g. Wash the membrane with TBST three times for 5 minutes each.
h. Incubate the membrane with the primary antibody and shake it overnight.
i. Wash with 15 mL of TBST three times for 5 min each.
j. Incubate the membrane with the secondary antibody and shake for an hour.
k. Wash with 15 mL of TBST three times for 5 min each.
l. Use the XRS Chemiluminescence imager to capture the image.
5 Antibactirial test
a. Add 200 μL bacterial suspension (E.coli or P.acnes) in each well of a 96-plate and then add 50 μL antimicrobial peptides of different concentrations.
b. Set blank control.
c. Culture bacteria for 24 hours.
d. Observe and test the antibacterial property with a microplate reader.
6 Large scale His-Tag fusion protein purification under nature conditions
( 1 ) Pretreatment before purification
a. Grow 1 L culture.
b. Induce at the temperature of 45 degree.
c. Spin cell culture 10 min at 6000 rpm at 4 centigrade, and discard supernatant.
d. Resuspend culture at 4 centigrade very gently with cold PBS buffer. Aliquot as following: 1 tube with 50 mL suspension (it means 500 mL original culture).
e. Incubate 30 min on the ice in the presence of 1 mM PMSF and 1mM Lysozyme.
f. Ultrasonic 3 cycles at 400 W power (one cycle is about 15 times, 10 seconds per time, 10 seconds apart).
g. Spin the cocktail for 60 min at 10000 rpm at 4 centigrade, collect supernatant, and take 40 µL of supernatant for PAGE-SDS.
( 2 ) Purification by nickel column
a. Connect the preinstalled nickel column to the high and medium liquid chromatography unit, avoiding bubbles.
b. Equilibrate nickel column with PBS buffer at the rate of 2 mL/min. Equilibration is confirmed by measuring pH and conductivity. Highest flow-rate and pressure limit: depend on the commercial resin.
c. Load supernatant which has been sterilized with 0.45 µm filter on the equilibrated nickel column at a low flow rate (2 mL/min).
d. Protein is eluted by step-gradient, using an elution buffer: 500 mM imidazole.
e. Low concentration imidazole steps at lower flow-rate (start collecting fractions) to eliminate low binding contaminant proteins.
f. Short gradient up to 500 mM imidazole at a very low flow-rate in order to obtain sharp peak, according to 280 nm optical density, 280 nm optical density, collect smaller fractions.
g. Samples from each step and each fraction are analyzed for protein content by SDS–PAGE. Protein-containing fractions are then pooled according to the profile obtained.
h. Optimize next purification and scale-up, according to results (optimization by adding, changing or eliminating steps).
i. After purification, equilibrate nickel column with PBS buffer. The nickel column was liquid sealed with 20% ethanol solution for long-term storage and reuse.