Good day, I performed western blot but I couldn't observe any bands of interest due to the high background. I saved the PVDF membrane just in case I would end up using it eventually and let it dried out for 1 month. We decided to stain the PVDF membrane with Poncea S and let it incubating for around 3 hours in a shaker. After washing with distilled water a few times, bands began to appear. However, the results are strange:
-There were 4 prominent bands across the area of the 60 kDa weight. It looks like the results you see when you are going to detect your bands using chemiluminescence.
- I can see faint bands across the membrane however, they begin to start at around 80 kDa and end at around 50 kDa or so.
What happened to the other bands? Isn't the membrane completely stained with bands and basically only the middle portion of the membrane has bands?
Important notes are:
-I had 4 samples that I used to run my gels
-Parameters that I used:
Electrophoresis: 90V for 15 minutes and 160V for 1 hour and 15 minutes.
Transfer: 0.4 amps for 1 hour and 15 minutes at 4 degree Celsius.
-The protein of interest that I was looking for was phosphorylated PKR, which weights at around that area
- I did not stripped the membrane of the antibodies
-I blocked the membrane with 5% BSA
Ponceau needs to be used before blocking and incubating the membrane, otherwise, you'll get a very high background. The purpose of Ponceau staining is to verify correct transfer of proteins from the gel onto the blot (10.1016/0003-2697(86)90263-0). You can verify correct electrophoresis by reversible gel staining procedures for example with zinc/imidazole (doi:10.1007/978-1-4939-8745-0_17).
Once you know that the electrophoresis and blotting have worked, you can block the membrane for example with 5% non-fat milk powder in Blotto, this will also remove the Ponceau. Then you start with the immune detection.
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