I have problems with my western blots in that I get no signal. I have tried all the usual changing of ab concentration, different blocking etc but nothing is working. Is it possible that my protein becomes miss-folded during sample preparation which then hides the epitope that the antibody recognises (and there is only one antibody available so not possible to change). if so what can be done about it.
In principle you should use denatured protein in western blots. because SDS/PAGE implies denaturation. You need an antibody that recognizes its epitope upon complete denaturation of the antigen. But sometimes, particularly if samples are not protected after reduction, S-S bonds can refold either in the native way or to give rise to missfolded proteins. This could affcet the result.
Are you sure the antibody you are usnig has been already tested for WB? If so, did you do the WB in the same cnditions already described for this antibody?
Check interaction of your AB an pro before western blot if it is possible.
When you run a gel your pro becomes liner, so AB that used for western detecting liner anti gene so I think your idea is wrong.
Have you ever had a successful Western blot with this antibody and a similar sample before? Sometimes the affinity of an antibody is insufficient for Western blotting.
Have you tested that your secondary antibody-enzyme conjugate retains enzymatic activity? This can be done by diluting it and mixing the dilution with the substrate.
Are you using the right substrate and buffer for the conjugate? (For example, you should not use PBS with alkaline phosphatase.)
Do you have a positive control on your blot, such a lane with the purified antigen?
If you have a polyclonal antibody specific to your target protein you should try immunoprecipitation and immunoblotting.
Article Immunoprecipitation-Western Blot for Proteins of Low Abundance
If not and if nothing else works I would think about far-western-blotting.
Article Detection and Quantification of Protein–Protein Interactions...
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