No signal of my normaliser, Western blot

  • Thread starter mountain
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Dear everybody,

Please help me out.

I am doing an experiment where i use B actin as the normalizer. The problem is after i have stripped the filters then some of the wells did not have the B actin signals. Even if the intensity of the target protein in that well had the same strength as the others, so this could not be a loading problem, could it? What is wrong? I bought the mouse monoclonal antibody against human B actin from Santa Cruz Biotechnology, Inc. Has anyone used this before? I used 500X dilution. Should i reduce the dilution factor? Do you know any well known manufacturers for selling good antibody against human B actin?

Thanks!
 

Moonbear

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I don't know if there's a problem with your beta-actin detection method (anything from a bad antibody to a bad method), a problem with damaging/washing off the beta-actin when you strip the antibody off, or if your samples are truly variable in the amount of beta-actin in them (this is why you need to choose the control carefully for normalizing; you could be loading unevenly or there could be variation in beta actin in your samples, which would indicate it's not a good protein to normalize to in your experiments).

The first place I'd check is whether it's in the stripping step or antibody. Try detecting beta-actin first, before the other protein of interest, and see if you still get the variation. If you do, it's not the stripping but something else, but if you don't have the same problem, then you need to troubleshoot your stripping step.

The other thing you can do, and should have started with, is a dilution series with your antibody to determine the optimal dilution for your system. If you're detecting your protein in some lanes but not others, then I wouldn't suspect the antibody.

Have you confirmed that you're running the gels an appropriate time and with proper acrylamide content to detect both your protein and the beta actin? Could your beta actin still be trapped in the loading gel (especially considering your other post asking about uneven running of gels)?
 
what are you using as a stripping buffer? glycine at pH 3?

personally, i would not strip to detect the actin unless you absolutely have to.
 
Thanks for the replies.

I have experienced that after stripping then one of my filters lost all of the signals even the MW marker. I wonder if stripping can do this as a side effect? Yesterday i stripped again and then reprobed with B actin and this time i have got the signals back. Have you experienced this?

Thanks again!
 

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