We are facing a problem during western blotting experiments in which some bands are disappearing or showing very weak signal. To troubleshoot this we have checked every possible factor possibly responsible for this fault like acryl amide/bis-acryl amide solution, buffers, transfer cassette, power pack, protein estimation methods (Bradford and BCA methods), protein transfer timings (1.5 h at 90 v, 2.5 h at 20 v and overnight at 10 v) etc, however unable to resolve the problem. As one can see in the figures (target protein GAPDH, Fig 1-5), we have loaded same samples on either side of the ladder and same amount of protein but few bands are missing or showing less expression (encircled with red colour). Kindly put your valuable suggestions.
1º antibody: ITT5052, Immunotag, 2º antibody: 7076S, Cell Signaling Technology
Hi Santosh
Previously, I have the same issue when I use the ECL substrate and film to exposure the WB. I do no know whether you also use ECL. In my case, I found that the band is missing because the substrate on this area is quickly being finished.
To solve this problem:
1. Dilute more of your primary antibody.
2. Use weaker ECL substrate.
3. Change your blocking buffer.
From your WB, it seems the antibody dilution should be fine. So, maybe change the blocking buffer, use some commercial one. I tried and found it works.
Hope this will help.
Best
Zeli
You have to exclude all factors individually, one by one, using a positive control first. Load the exact same sample into all wells, then run the blot. If you get this phenomenon, then it is not the samples, but something of the reagents/materials.
You didn't mention the membrane (white paper). Is it old? Freshly bought? Do you wear gloves when handling it? Is it always immersed in liquid (i.e. you don't allow it to dry)?
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