Western experimental procedure
Sample preparation
1. Protein extraction
Equipment needed: homogenizer, RIPA lysis buffer, PMSF (protease inhibitor), phosphatase inhibitor, scissors, tweezers, disinfected alcohol swabs, PBS, pipette, centrifuge.
Lysis buffer preparation – RIPA lysis buffer: PMSF = 100:1.
1) Tissue processing:
First, disinfect the scissors and tweezers with alcohol wipes. Take an appropriate amount of tissue and place it in a homogenizer. Use scissors to chop the tissue and add an appropriate amount of lysis buffer and ice water to homogenize for 1-4 minutes. After homogenization, transfer the solution to an EP tube and store it for later use. (Tissues with more blood can be washed with PBS before homogenization.)
2) Cell processing:
A. Treatment of adherent cells: Remove the medium, wash with PBS, add an appropriate amount of lysis solution to lyse the cells, and pipette appropriately to dislodge the cells. Large bottles of adherent cells can be washed down with trypsin first and then lysed. The lysis method is similar to the treatment of suspended cells.
B. Treatment of suspended cells: Suspended cells are first placed in an EP tube and centrifuged at 3000 rpm for 5 min to remove the supernatant medium. 100 μL of lysis solution is added to every 20 μL of cells and the mixture is repeatedly pipetted until the cells are completely broken.
Centrifuge the treated sample at 12,000 rpm for 5 min, and take the supernatant to obtain the extracted protein. It can be stored at 4 °C for one week, at -20 °C for about 2 months, and at -80 °C for half a year.
2. Protein concentration determination:
enzyme-labeled plate, enzyme-labeled instrument, centrifuge, 1 mg/mL BSA protein standard solution, BCA reagent, PBS.
1) Standard curve: Use a solution of 20 μL total volume mixed with BSA and PBS to make a standard curve. The specific operation is as follows: dilute the BSA protein standard according to the contents of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 dilute the BSA protein standard, and make a replicate well for each concentration (0, 4 μL, 8 μL, 12 μL, 16 μL, 20 μL BSA, the rest is made up to 20 μL with PBS).
2) Sample dilution to measure protein concentration: 10-fold dilution method: Take 18 μL PBS + 2 μL sample supernatant (supernatant after centrifugation at 12,000 r/min for 5 min for the sample processed in the previous step) and make two replicates for each sample.
20-fold dilution method: Take 19 μL PBS + 1 μL sample supernatant and make two replicates for each sample.
Generally, 10-fold dilution is suitable for cell proteins and 20-fold dilution is suitable for tissue proteins, with a total volume of 20 μL.
3) Preparation of BCA reagent: BCA A solution: B solution ratio is 50:1. This reagent is prepared fresh and used immediately. Take care not to be contaminated with other proteins.
4) After the standard curve and the sample are added to the wells, add 200 μL of BCA mixing reagent to each well and place it in an incubator at 37 °C for 15 min. The OD value is measured at a wavelength of 568 nm using an enzyme labeling instrument. The measured OD values of the standard curve are used to obtain the formula of the standard curve by taking the average of the concentration gradient (two wells) and subtracting the blank. The OD value of the measured sample (three wells) is averaged and the protein concentration is obtained by calculating according to the formula of the standard curve. The sample loading amount is calculated according to the standard of 50 μg/sample loading hole, and the calculation formula is: sample loading amount = 50/protein concentration. Note: All obtained OD values must be subtracted from the background (OD value of the blank well in the standard curve) before calculation.
3. Sample processing:
Protein samples: Mix 5×SDS loading buffer with boiling water at a ratio of 4:1 for about 10 minutes. Samples with too high a concentration can be diluted with PBS and then boiled. After boiling, the samples can be stored at -20 °C for later use (can be stored for half a year). For long-term storage, the samples must be stored at -70 °C.
Experimental procedure
1. Gel preparation
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1) Prepare the separating gel in proportion according to the molecular weight of the target protein, mix slowly to avoid mixing in oxygen due to vigorous mixing. After the gel solution is mixed well, slowly inject the mixed gel solution into the two glass plates, and then carefully inject a layer of anhydrous ethanol on the surface of the liquid to prevent oxygen from contacting the gel solution and affecting the polymerization of the gel. Keep it level and let it stand in a 37 ℃ incubator for about 1 hour until the gel solution completely polymerizes and solidifies.
2) Prepare the concentrated gel solution according to the ratio, mix slowly, discard the upper layer of anhydrous ethanol, and absorb the anhydrous ethanol on the solidified gel with filter paper. Gently inject the concentrated gel solution, insert the comb carefully to avoid bubbles at the tooth tips. Leave it in a 37 ℃ incubator for about 1 h until the gel solution completely polymerizes and solidifies. Carefully pull out the comb and prepare to load the sample.
2. Sample loading
Add electrophoresis buffer to the electrophoresis tank. The electrophoresis liquid on the inner side of the two glass plates should be higher than the sample loading hole, and the electrophoresis liquid in the outer electrophoresis tank should immerse the bottom of the gel. The liquid level on the inner side of the glass plate should be higher than the outer side. Calculate the sample loading volume (the treated sample, pre-stained protein marker), to ensure that the total protein volume in each well is 30-50 μg, and that the total sample volume is less than 30 μL. Pay attention to keep the loading time as short as possible to avoid sample diffusion.
3. Electrophoresis
After loading the sample, close the lid of the electrophoresis tank, pay attention to the positive and negative electrodes, and select an appropriate voltage for electrophoresis. In a discontinuous system, the electrophoresis voltage of the upper concentrated gel (recommended 80 V) should be lower than that of the separating gel (recommended 110-150 V) to achieve better concentration. The sample will be compressed to the same horizontal line when it enters the separating gel. Electrophoresis until the bromophenol blue dye front reaches the end of the gel and stop electrophoresis. The electrophoresis time is about 2-3 hours.
4. Electrophoresis (wet transfer method)
1) After electrophoresis, remove the gel and rinse it in electrophoresis buffer (chilled) for a few seconds. Open the electrophoresis transfer device in sandwich mode, place a special sponge soaked in transfer buffer on each side, then place a protein transfer pad (qualitative filter paper) soaked in transfer buffer on each side. Place the gel flat on the filter paper on the cathode side . Finally, place a PVDF membrane soaked in transfer buffer and pre-soaked in methanol on top of the gel, remove any air bubbles, clip the electrotransfer clamp shut, and make sure that there are no air bubbles between the PVDF membrane and the gel, between the PVDF membrane and the filter paper, or between the filter paper and the gel.
2) Fill the transfer tank with transfer buffer, insert the electrotransfer clamp, and place the transfer tank in ice water to ensure that the PVDF membrane is close to the positive electrode and the negatively charged amino acids and proteins migrate towards the positive electrode.
3) Select constant current for membrane transfer. The recommended current for each transfer tank is 150-300 mA, and the time can be adjusted appropriately based on the molecular weight of the target protein band.
4) After the transfer is complete, place the PVDF membrane in the leuco red staining solution for 5-10 minutes at room temperature, and red bands can be seen. The red bands can be washed off with washing buffer several times for subsequent experiments.
5. Blocking
1) Rinse the blot membrane: rinse appropriately at room temperature using washing buffer.
2) Remove the washed membrane and place it in blocking buffer (5% skim milk or 5% BSA), shake in a shaker and block at room temperature for 1.5-2 hours.
6. Incubation of antibodies
The indirect method is mainly used, that is, unlabeled specific primary antibodies are first added, and the antibodies bind to the antigenic proteins on the membrane. Then, enzyme-labeled (peroxidase or alkaline phosphatase) secondary antibodies are added for detection.
1) Diluted primary antibody is added to the blocked membrane and incubated overnight at 4 °C.
2) The membrane is washed with washing buffer for 10 minutes × 3 times.
3) Diluted enzyme-labeled secondary antibody is added and incubated at room temperature for 1 hour. The membrane is washed 3-6 times for 10 minutes each time.
7. Detection
The detection method varies depending on the marker of the secondary antibody. Common detection systems include chemiluminescence (ECL) and the DAB chemiluminescence detection system.
Chemiluminescence (ECL):
1. Using HRP to catalyze chemiluminescent substances generates an unstable intermediate substance, whose decay produces a visible chemiluminescent band in a dark room.
The result is recorded using the X-ray film sensitization principle.
2. Preparation of ECL chromogenic reagent: Mix chemiluminescent substrates A and B in equal volumes according to a 1:1 ratio.
3. Cover the blot membrane with the mixed chromogenic substrate for 1-5 minutes and observe the fluorescence effect under dark conditions.
4. The result can be recorded in a darkroom using a radiographic self-developing film or a chemiluminescent imaging system.
5. If using autoradiography film, expose for a few seconds to several minutes, control the exposure time according to the fluorescence intensity, and determine the correct exposure time for the measured antigen based on the development effect. After exposure, the film is first soaked in the developing solution until bands appear, and then placed in the fixing solution to fix the image. Finally, the film is rinsed with water and hung to dry.
DAB detection:
1. Mix 1 mL of water with one drop each of developers A, B, and C.
2. Color development: Spread an appropriate amount of DAB developer evenly over the hybridized membrane and leave it at room temperature to observe. Obvious brown-yellow protein bands may appear.
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