I am getting multiple bands for TNF-alpha and IL-6 on western blot membrane which make me difficult to search for the desire band. is it due to problem with the primary antibody or improper washing?
Hello Imdad,
Maybe you can post a picture of your blot, that will make it easier to help.
Multiple bands on a western may have several reasons. It may be a "babd" Antibody, i.e. it will unspecifically stain other proteins. May may be able to reduce this effect by diluting your primary AB further. Also Blocking may be an issue. There may also be several isoforms of your protein, or split variants, that will serve as specific antigene - that should be in the datasheet, but it may still not.
Hope that helps.
Hey buddy, I barely see anything in the picture. I don't know if the exposure it's not really good or what. I can appreciate some bands but I still don't know the antibodies that you are using.
IL6
Now I get to see the bands properly. Still don't know what those bands are. To me it looks like something non-specific, but it's hard to know.
It looks like it has nothing to do with the concentration of the antibody that you're using, since all bands more or less have the same intensity. I agree with Francisco that it looks non-specific binding (due to blocking?). It could also be an IL6 isoform or glycosylated form (see the website of abcam for example: http://www.abcam.com/il6-antibody-ab6672.html#description_images_1). You could add a positive control to your blot if you haven't done this already.
What sample did you run? (cell lysate or supernatant) TNF-a and IL-6 is secreted protien, ELISA is much easer than WB for detection of them.
To add to what the others said, It might be useful to run a positive control. Either your the recombinant proteins or cells spiked with them. Also I have to agree with Jargalsaikhan Dagvador that you may be looking in the wrong place.
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