Hi,
After immunoprecipitation and elution, I check the eluate with western blot. If I incubate the blot ONLY with secondary antibody, it gives the same signal as incubatin with primary & secondary?
Is it because the secondary is "sticky" and will bind to membrane-rich patches in the membrane?
How to validate if I immunoprecipitated the protein, if the secondary also gives a signal without the primary?
Thanks a lot!
Hi,
there could be multiple reasons for your observation.
- trivial question: wrong antibody? degraded/inactive antibody? (use positive control, every time, if possible!)
- "wrong antibody" could also mean wrong for Western Blot! If it works for IP it doesn't mean it has to work for WB (and vice versa), so try at least one or more different antibodies
- Concentration of your antigen too low or primary antibody with low affinity (increase antibody concentration or load more sample but don't overload the gel lane)
- Concentration of secondary antibody too high, therefore high background (try lower conc., some secondary antibodies can be used in really high dilutions with a final conc. of ~10ng/ml)
- Combination of second and third option is even worse, you need to find the right dilutions for both antibodies (don't forget the positive controls!)
Hi there,
So the secondary gives you non specific signal only as it is independent of the presence of the primary. As a consequence your primary does not seem to bind the membrane...
Hi again, your secondary antibody has no specificity for your protein of interest so the band you see is certainly non specific. Spotting a band at the right size on a membrane doesn't mean it corresponds to the expected protein.
What is the specificity of the secondary antibody and what is your protein of interest?
Is the band intensity exactly the same when using either both or secondary antibodies? (any picture?)
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