For my western blot, I transferred at 80v for 70 minutes, but after I stained my two gels with coomassie, I was able to observe some residual protein at mid to large size despite good transfer of my ladder. Usually I transfer at 23v overnight. In other words, is it normal to have some protein left behind in the gel and will this affect the relative levels of proteins immunodetected between samples if transfer is not 100% complete? In other words, if there are differences in my protein of interest between control and treatment and transfer is partially incomplete, will I have a chance of missing any possible differences in protein levels between them? Has always been a lingering question for me.
In my opinion, there is no problem at all. If you have any concerns, you can set two or three repeats by changing the order of your samples. These results will be more convicing.
The important thing is to be consistent. Always use the same transfer protocol and fresh transfer buffer when repeating the experiment if you need to compare the signals for different proteins on the blot. Of course, it is also important that the antibody quality and blotting protocol be consistent as well.
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