Hi,
I am trying to analyze the protein expression of 2 different proteins via western blot. Since both are different isoform I am using two different antibodies for each respectively. However, in result I can visualize a clear blot with only 1band in one blot while for other blot the band are detected like a ladder (its high background or the antibody is detecting every single band?). I can think of is that it has to do something with the antibody only.
Therefore, could anyone tell how to improve the background or share some possible options one can try?
One reason you may be seeing a ladder with one of the antibodies, if it is an antiserum instead of a monoclonal, is that the antiserum contains antibodies that react with other proteins in the sample. To improve the specificity of the antibody, you can pre-adsorb it with a cell extract that does not contain the antigen of interest.
Another possible reason for seeing a ladder is degradation of the antigen. Including protease inhibitor cocktail when preparing the cell extract may help reduce this problem.
A third reason for a high background is that the antibody concentration used to probe the blot is too high.
Thank you Adam B Shapiro for the valuable suggestions, I have tried already the second and third possible reason suggested by you with no good outcome, I would therefore try to proceed with the pre-adsorbed antibody method.
It could depend on the antibody as well as if the blocking reagent is not working proper , if the antibody is polyclonal this isue exists also how is the diference between this proteins with respect to size and structure , it all matters , try using monoclonal antibody and also the blocking reagent and the duartion you are keeping for blocking
You may consider these few suggestions to improve your background.
1. Reduce the amount of primary and secondary antibody: Decreasing the amount of primary and secondary antibody used in the Western blot can reduce background.
2. Block non-specific binding sites: Non-specific binding of the primary and secondary antibody can contribute to background. Blocking these sites with a blocking buffer such as BSA or non-fat dry milk can reduce background.
3. Wash the blot thoroughly: Excessive washing of the blot can remove the primary and secondary antibodies and reduce background.
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