Team:GreatBay United/Measurement
Measurement
Experimental Process
In our experiment, we included three systems:detection system,cascade system and agglutination system, in which HCV protease is the key protease connecting the cascade system and the aggregation system. We successfully expressed this enzyme. In order to test the activity of this protease, we synthesized a polypeptide fluorescent substrate for this enzyme recognition cleavage check point. This substrate is: (amino acid sequence). At both ends of it, we added the modifying group DABCYL/Glu (EDANS). When the substrate peptide chain is cleaved, the two groups separate and fluoresce. This fluorescence detection condition is ʎex/em 340 and 490nm. On this page, we will introduce in detail the method of using fluorescent polypeptide substrates to detect enzyme activity, and provide our summarized experiences gained during this experiment process.
1.Fluorescent substrate synthesis
We choose Kingsley's company for synthesising fluorescent substrate.Fluorescent substrate synthesis requirements: purity > 85%, endotoxin content needs to be controlled, total mass 200mg.
2.Preparation of substrate solution
Polypeptide fluorescent substrates are often dissolved using DMSO. We formulated a 100 μM mother liquor. In subsequent experiments, the mother liquor was diluted to obtain different concentrations of substrate solutions. After the substrate solution was successfully prepared, it was aliqued into 1 mL/tube and stored to -20 ℃.
3. Protease reaction buffer
Protein reaction buffer: 200 mM Tris-HCl, 200 mM NaCl, 200 μg/mL BSA pH 8.0 We preset the whole reaction system to 200 μL. Due to the need to adjust the proportion of different components in the experiment, we formulated the buffer mother liquor. The HCV protease activity assay conditions are provided in the references (50 mM Tris-HCl, 50 mM NaCl, 50 μg/mL BSA pH 8.0).This buffer is added to the system at a ratio of 4X when used, and the protein reaction buffer mother liquor is successfully prepared and stored to 4 ℃.
4. Use other materials and testing instruments
This experiment uses Corning Incorporated enzyme labeling plate. This enzyme labeling plate is a black plate and CLear bottom, because this enzyme labeling plate can prevent signal interference between adjacent samples. The Tecan Infinite 200Pro model enzyme labeling instrument was used for detection. The detection condition is ʎex/em 340 and 490nm.
Testing process:
Experimentally, we tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of either HCV protease or substrate concentrations.
1. Different HCV protease concentrations
In this group of reactions, the total reaction system of each sample is 200 μL, including 50 μL protease reaction buffer, a final substrate concentration of 5 μM(substrate concentration is 100 μM, and the addition amount is 10 μL), HCV crude enzyme solution addition amounts are 0 μL, 5 μL, 30 μL, 50 μL, 100 μL, 140 μL respectively.The rest of the system is supplemented with ddH2O(the amounts are140μL、135μL、110μL、90μL、40μL、0μLrespectively). In order to eliminate the influence of substances added to the reaction system on the data, we designed a treatment group for each samples. In the treatment group, an equal volume of DMSO (solvent of the substrate) was used to replace the substrate in the experimental group. The rest of components remain unchanged.
2. Different substrate concentrations
In this group of reactions, the total reaction system of each sample is 200 μL, including 50 μL protease reaction buffer, 5 uL HCV crude enzyme solution, the final concentrations of the substrate are 0 μM, 5 μM, 25 μM, 50 μM, 150 μM, 250 μM respectively, increased sequentially.In order to control the overall product of the substrate unchanged (10 μL), it is necessary to prepare different concentrations of the substrate solution (substrate concentrations are 100 μM, 500 μM, 1 mM, 3 mM, 5 mM), and uses 135 μL ddH2O to make up the total reaction system to 200 μL.In order to eliminate the influence of substances added to the reaction system on the data, we designed a treatment group for each substrate concentration sample. In the treatment group, an equal volume of DMSO (solvent of the substrate) was used to replace the substrate in the experimental group, and the rest components remain unchanged.
3. Detection conditions
First,add different experimental samples to the enzyme labeling plate,then directly put the enzyme labeling plate into the enzyme labeling instrument without covering, and set the reaction program. a.The reaction temperature is 37 ℃ ± 0.5 ℃, until the temperature reaches the requirements to start the reaction. b. Read the data, ʎex/em 340 and 490nm, before reading the data, shake for 10s. c. Read the data every 5min for 70 cycles in total. d. Read the data, ʎex/em 340 and 490nm, before reading the data, shake for 10s. e. Read the data every 30 min for 10 cycles in total. After the measurement, analyze and process the experimental data.
The experience we summarized during the experiment
1. There are many test samples in the experiment. When the components of multiple samples are similar, you can prepare a mixed solution and then repackage it. In this part of our experience, we need to prepare 30 samples that are mixed. It is best to configure the mixed solution with 35 samples to ensure sufficient use. 2. When the substrate mother liquor is not easy to dissolve, you can consider accelerating dissolution in a water bath pot. 3. In the reaction program, we can adjust the "gain" value of the program and correct the result to ensure that the fluorescence signal intensity will not exceed the range(otherwise it would show "OVER"), and "gain" is set to 50 in our experiment.
Analysis and dealing of experimental data
Experimentally, we tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of either HCV protease concentrations.The results showed that the reaction trends of different groups is the same, and the fluorescence signal intensity is positively correlated with the enzyme amount at the initial stage. When the substrate is completely reacted, the fluorescence signal intensity tends to be consistent.
We also tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of substrate concentrations.The results showed that the fluorescence signal intensity of the reaction has the same change trend. When the substrate is completely reacted, the higher the substrate concentration, the stronger the fluorescence signal intensity.
In this experiment, fluorescence modified peptide substrate was successfully used to test the enzyme activity of HCV, and Mie's equation was calculated by mathematical modeling analysis.