We used tumor and normal tissue lysate and an equal amount of 30 ug of protein was loaded to each well. The lysate was stored in -80 degree. After mixing the dye it was stored in -20 degree. Also we observed a high non-specific background along with intense multiple bands below 55 kDa. We blocked the membrane with 5% Skimmed milk in TBST and also 5% BSA in TBST the next time. The background issue is still not solved. Other proteins show crisp bands with very light background. Should we change another loading control?
Hello Shivaani Srinivasan
You could change to another loading control. But I would like to mention two possible reasons for this type of a problem.
1. The primary antibody for alpha-tubulin may not be of a good quality. Use a high-affinity primary antibody that can bind and detect its antigen at a low working concentration. A low-affinity antibody will require a higher working concentration. As a result, higher working concentrations increase the likelihood of off-target binding and high background.
2. If you freeze-thaw your samples several times, there is a possibility that alpha tubulin detection could have become problematic due to degradation. May be alpha tubulin could be less stable than other proteins.
Best.
Was it native WB or denatured WB? If it was denatured one how did you prepare you sample?
-20 isn't ideal for sample storage, -80 is (for fresh frozen sample). If you are doing a denatured WB and when you say "mix the dye" means "mix the lysate with sample buffer and cook for denaturation", then you can keep the denatured sample in 4 celsius frig for a couple of weeks for no problem.
Yi-Chun Cheng we did denatured PAGE. We stored the denatured sample at -20 degrees though.
Laurence Stuart Dawkins-Hall
thank you for your answer. Which loading control would you specifically suggest for tissue lysates?- Badges
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