1. LB Broth Medium and Antibiotics:
- Prepare LB broth medium based on the "Molecular Cloning Laboratory Manual" (by J. Sambrook and D.W. Russell). For every liter of medium, add 10g of tryptone, 5g of yeast extract, and 10g of NaCl to 950mL of deionized water (make up to 1L). For solid medium, add an additional 15-20g of agarose, then autoclave at 121°C, 0.1Mpa for 20 minutes.
- Prepare a stock solution of ampicillin: dissolve 50mg of ampicillin in 1mL of sterile broth. When using, add 100μL of this stock solution to 100mL of medium.
- Prepare IPTG stock solution: dissolve 1mol of IPTG in 1mL of sterile water. When using, add 10μL of this stock solution to 100mL of medium.
1. Take BL21(DE3) competent cells from -80°C and quickly thaw them in ice (100 μL). Add 10 μL of target DNA. Gently mix by tapping the bottom of the EP tube (avoid pipetting), and let it sit on ice for 30 min.
2. Heat-shock the tubes in a 42°C water bath for 60 s, then immediately transfer to an ice bath for 3 min (avoid shaking the tubes as it may reduce the transformation efficiency).
3. Add 500 μL of antibiotic-free sterile broth (LB) to the tubes, mix, and then incubate at 37°C, 180 rpm for 60 min for cells recovery.
4. Centrifuge at 5000 rpm for 1 min, retain 100 μL of the supernatant, gently resuspend the cell pellet, and plate it on LB agar containing ampicillin.
5. Invert the plates and incubate overnight at 37°C.
1. Transfer a portion of the grown cells at a 1/100 ratio to 500 mL expression medium (LB). Incubate on a shaker at 37°C, 220 rpm. Measure the OD600nm hourly. After the first hour, measure every 30 min and save 1 mL of culture each time.
2. When the OD reaches 0.6-0.8, add IPTG.
3. For IPTG: dissolve 0.0715 g of IPTG in 1.5 mL of sterile water. Add 500 μL to each of the 500 mL culture flasks.
4. Continue incubation for 12 h.
1. Transfer the culture to centrifuge tubes and centrifuge at 10000 ×g, 4°C, for 3 min to pellet the cells.
2. Resuspend the pellet in lysis buffer at a ratio of 10 mL buffer per 1 g of wet cell weight.
3. Let the mixture sit on ice for 30 min.
4. Lyse the cells using an ultrasonicator (50% power; 3 s on, 5 s off, for 15 min).
5. Centrifuge the lysate at 10000×g, 4°C for 15 min. Discard the pellet and filter the supernatant using a 0.45 μm filter.
1. Equilibrate the column with 3 column volumes of deionized water.
2. Balance the chromatography column with equilibration buffer (3-5 column volumes).
3. Load the filtered lysate onto the column.
4. Wash away non-specifically bound proteins with equilibration buffer (3 mL, 7 times).
5. Elute the target protein with elution buffer (3.5 mL, once).
6. Clean the column with elution buffer containing 500 mM imidazole (3-5 column volumes).
7. Rinse the column with deionized water (8-10 column volumes).
8. Store the column in 20% ethanol (3-5 column volumes) and seal. Keep at 4~8°C.
1. Clean the desalting column with 4 column volumes of deionized water.
2. Equilibrate the column with 4 column volumes of desalting buffer.
3. Load 1.5 mL of the protein solution onto the column.
4. Elute the protein using 2 mL of desalting buffer.
5. Equilibrate the column again with 3-5 column volumes of desalting buffer.
6. Repeat the desalting process for the next sample.
7. Clean the desalting column with 3-5 column volumes of deionized water.
8. Store the desalting column in 20% ethanol (3-5 column volumes) and seal. Keep at 4~8°C.
Prepare 10 mL of 10% separating gel (as per table). Mix immediately and pipette slowly between the glass plates from the top-left corner, filling until about 1.5 cm from the top edge.
- Ensure no leakage; if so, stop and re-seal the mold.
- Act quickly during gel preparation; add TEMED just before pouring. Pour slowly to avoid bubbles.
- A clear interface between the gel and the water indicates good polymerization.
Carefully add a layer of distilled water on top of the separating gel. Leave it vertically at room temperature for around 30 minutes. Once the gel is set, remove excess water with filter paper. Note This step ensures an even gel surface and helps in the polymerization by excluding air.
Prepare 5 mL of 5% stacking gel (as per table). Pour the gel until it reaches the edge of the front glass plate. Note: Ensure no bubbles at the comb's insertion point and that the comb's bottom is level.
After the gel sets, remove the glass plates. Carefully remove the comb and the sealing tape. Place the glass plate with the thinner one facing inward on the electrophoresis frame. Fill the tanks with electrophoresis buffer. Note: Handle the glass plates with care to avoid breakage.
a. Mix 10 μL protein sample with 10 μL of sample buffer. Heat in boiling water for 3-5 minutes and let it cool to room temperature.
b. Load 10-15 μL of the sample, ensuring no dispersion.
c. Load 5 μL of protein marker. Note: Remove any air bubbles in the wells and avoid piercing the gel with the pipette.
Connect the electrophoresis device: the anode to the positive terminal and the cathode to the negative. Start electrophoresis at 80 V, then increase to 120 V once the samples enter the separating gel.
Stop when the bromophenol blue band is about 1 cm from the gel bottom.
Remove the securing plate, separate the glass plates, and cut off the stacking gel.
Mark one end of the gel. Note: Handle the gel delicately.
Immerse the gel in Coomassie blue and stain overnight.
After staining, pour off the stain, briefly rinse the gel, capture the image, and record the results.Add reagents to a 96-well plate as mentioned in the table.
Mix well and measure absorbance at 595 nm. Plot a standard curve.
Mix sample with G-250 solution. After 2 minutes, measure OD595.
Calculate protein concentration and recovery rate using the standard curve.
1. Mix 3 mL of pH 7.4, 1% soluble starch solution with 2 µg of commercial α-amylase in a centrifuge tube.
Immediately start the reaction in the spectrophotometer.
2. For another sample, mix reaction buffer (with 20 mM substrate starch concentration) with a pre-filtration protein sample. Shake vigorously and start the reaction in the spectrophotometer.
3. Continuously measure absorbance at 250 nm for 180 seconds. Analyze the rate of change of Abs vs. time.
4. Calculate enzyme activity, turnover number, specific activity, purification fold, and yield from the linear section of the curve.
1. Add buffers, substrate, and enzyme solutions to eight centrifuge tubes as described. React in a 25°C water bath for 3 minutes, add the colorimetric agent, and boil for 5 minutes. After cooling, start the measurement using a microplate reader.
2. Calculate the reaction rate at different substrate concentrations. Perform non-linear regression using Graphpad Prism software.
1. Add 2.4 mL of reaction buffer at varying pHs (5.5, 6, 7, 7.4, 8, 9, 10) and a substrate concentration of 20 mM to seven centrifuge tubes. Then add 20 µg of enzyme.
2. Continuously measure absorbance at 250 nm for 180 seconds.
3. Calculate the specific enzyme activity at different pHs. Plot the results to determine the optimal pH for enzyme activity.