I am using β-actin as a loading control for my western blots to enable quantification of proteins. I never used to have an issue with this protein but recently I am getting faint bands during exposure - but not for all bands. I have tried varying exposure times and decreasing the volume of sample but I am still getting these faint bands. I can't find a pattern between samples which lead to faint bands. I normally probe for β-actin after stripping the blot twice but have tried probing for β-actin straight away also and still experience these bands. The bands I get for other proteins from these samples are fine so I am skeptical it is the samples themselves.
Any help on this would be greatly appreciated!
Thanks,
Richard
How long has the primary antibody been diluted, the antibody might have got degraded if already diluted and kept for a longer interval , also check for the mRNA expression of b actin , then may be you can decide wether to use this or some another protein as endogenous control
Trisha Chattopadhyay, The antibody has been used fresh each time and B-actin has worked fine for us before and here between blots, samples can be fine or faint, therefore I don't believe it is antibody degradation nor differential expression.
If the membrane is old, it may be less sensitive to the antibody. Try using a fresh membrane and check the storage conditions of the membrane.
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