Western blot (WB), also known as Immunoblotting, is a technique used to identify specific proteins from complex protein mixtures. Protein samples are separated by SDS-PAGE electrophoresis, transferred to a solid support (such as PVDF membrane or nitrocellulose membrane) using electric field, and then the target protein is detected through antigen-antibody specific binding and labeled secondary antibody color development.
Full Process Operation Video
Experimental Procedure
Sample Preparation
The protein sample preparation process is shown in the figure below:
After protein extraction, the concentration can be determined by BCA method or Bradford method to ensure stable and controllable loading amount.
After determining the protein concentration of each lysate, take equal quality lysate and add 4xSDS Sample Loading Buffer.
After mixing, boil at 95-100°C for 5-10 minutes to reduce and denature the sample. The lysate can be aliquoted and stored at -20°C for later use.
Note: For some membrane proteins, transmembrane proteins, and even nuclear proteins (Rb protein), high-temperature boiling may cause aggregation to form <span style="color: red">multimers or "ghost bands"</span>, leading to electrophoresis band shift or loss.
SDS-PAGE Electrophoresis
1) Load equal amounts of protein and protein marker into SDS-PAGE gel wells. The total protein loading for cell or tissue lysates is 10-40 μg, and for purified proteins is 10-100 ng.
2) Run the gel at 80V for 20-30 minutes, then adjust the voltage to 120V and continue running for 1-2 hours until the bromophenol blue band runs to the bottom of the gel (or adjust the running time according to the actual molecular weight).
Tips
- 1) Ammonium persulfate decomposes slowly and needs to be prepared freshly in time;
- 2) During electrophoresis, proteins not only diffuse and separate from top to bottom, but also diffuse and separate to <span style="color: red">both sides</span>, so add appropriate amount of 1x loading buffer (or irrelevant protein sample) to empty sample wells to prevent "smiling" bands;
- 3) Under the premise of not affecting the detection signal, try to load less sample, the resulting bands will be sharper;
- 4) Do not overheat during electrophoresis, overheating will cause band dispersion or even protein degradation;
- 5) Prepared gels can be stored in a 4°C refrigerator for about 2-4 weeks (prevent drying, prevent freezing and thawing).
Transfer (Take Wet Transfer as an Example)
1) Preparation: Soak PVDF membrane in methanol for 30 seconds, rinse twice with pure water, then soak in 1x transfer buffer (wet transfer). Soak filter paper and sponge in transfer buffer as well.
2) Remove gel: Take out the slide from the electrophoresis tank, remove the gel and soak it in transfer buffer.
3) "Sandwich" assembly: Arrange them tightly in the order of sponge → filter paper → membrane → gel → filter paper → sponge, ensuring no bubbles between each layer.
4) Constant current method (generally 200-300 mA, 90 minutes) or constant voltage method (generally 75V, 90 minutes) can be used for electrotransfer.
5) After transfer is complete, take out the membrane and stain with Ponceau S to confirm that the transfer is successful.
Tips
- 1) Try to clean the sponge thoroughly after each transfer; filter paper should not be reused as much as possible;
- 2) Transfer time needs to be optimized according to protein size, reference constant voltage for wet transfer of 20~250kD: 75V, 80-90 minutes;
- 3) Transfer buffer can be used at most 2 times to avoid excessive heat generation leading to protein degradation;
- 4) Wet transfer is recommended for large molecular weight proteins, semi-dry transfer may not necessarily improve transfer efficiency even if the time is extended (and may even lead to degradation);
- The transfer of protein Marker and target protein is not completely equivalent (common in semi-dry transfer), especially for high molecular weight proteins.
Antibody Incubation
1. Blocking: Recommended 5% skim milk (in TBST) for 1 hour at room temperature.
2. Primary antibody incubation:
1) Take the membrane out of the blocking solution and wash the membrane three times with 1x TBST, 5 minutes each time.
2) Dilute the primary antibody with primary antibody dilution buffer according to the recommended dilution ratio, recommended incubation at 4°C overnight.
3. Secondary antibody incubation:
1) Wash the membrane three times with 1x TBST, 10 minutes each time.
2) Use HRP-conjugated secondary antibody at the recommended dilution ratio (can be diluted with blocking buffer), incubate for 1 hour at room temperature.
Development
1) Wash the membrane four times with 1x TBST, 10 minutes each time.
2) During washing, mix ECL substrate A and B at 1:1 to prepare developing solution.
3) After fully reacting ECL substrate with the membrane, wrap the membrane with plastic wrap and fix it on the dark clip. Expose the membrane to dark photographic film or use a chemiluminescence imager to read the signal.
Tips
- The choice of developing solution is also very important, the laboratory should at least have two sensitivity levels of ECL. It is recommended to prepare the developing solution fresh, and when trying a new target for the first time, it is best to try with low sensitivity ECL first, and then change to high sensitivity ECL if it is weak.
- 1) When shooting with an imager, <span style="color: red">do not recommend</span> using the machine's <span style="color: red">auto-exposure</span> mode; it is recommended to shoot 5 or 10 images within a fixed time, and select the best image later according to the actual situation;
- 2) Imagers can generally adjust parameters such as image zoom, resolution/sensitivity.
If after initial exposure:
A. The band is very strong, after re-adding ECL, you can choose high resolution, low sensitivity mode;
B. Conversely, if the band is a bit weak, you can also try low resolution, high sensitivity mode.
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