I use the santa cruz C4 sc-47778 B actin anti-body as the reference in my blots.
When i load only 3 samples on my gel, the antibody seems to bind to something and it shows as if i loaded the whole gel (12 samples), i am pretty sure it is picking up the ladder as well which i found strange. Does that mean my antibody is infected?
i tried it multiple blocking buffers, higher concentration and even an overnight block and seemed to get the same thing.
Hello, yes it looks like your antibody is not specific anymore? I have a question though, are the bands detected from the samples you loaded different from the one you got from the empty wells? if it is the case then it might be your secondary antibody that is not specific too
Iskander Madhi hi iskander, thank you for your reply. The bands look exactly the same. the antibody i am using is a conjugated antibody so i just add that.
Basma Elbakary I think you should try with another antibody (B-actin one, or even another control protein) to check whether the reason is related to western blot procedure (running, gel, transfer, blocking..). If you have clean detection with other antibody then the problem is really originated from teh antibody you use now.
Good luck
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