I am trying to develop my western blot using an antibody targeting a 17kda protein. It is binding non-specifically and not giving any band(like in the photo). What does it mean is happening? How should i trouble shoot this?
My first guess would be a problem with your gel or your protein sample. If it was an antibody issue then you probably would have black all over the gel. But this seems fairly discrete as a single band. So maybe the underlying band is what is strange. You might try running a gel with the same samples and staining it to see how the protein bands look.
Or possibly just repeat the blot and try again.
What percentage of acrylamide did you use for your SDS-PAGE ?
Manita Raina I agree with Michael J. Benedik. Its just the shape of "band". Your sample has run along sides of well. This can happen for various reasons, including running the gel too fast (high current).
Is the picture showing the band you expected? Where is the Non-specific binding? Cannot see any other bands.
Numerous causes might be to blame for this kind of problem. What I can recommend is that you lengthen your blocking period to at least 2 hours and that you avoid incubating the secondary antibody for longer than 2 hours.
Use freshly prepared buffers and make sure about the proper transfer. Hope you will get a good result next time.
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