I have anti c3 primer antibody (abcam ab11887). It was incubated 1h room temperature and washed 3 times (10 min interval) TBST buffer, then I used second antibody 1h, washed 4 times. The membran included b actin was so clear, but c3's membrane was unclear (i added a photo). How long should I incubate? Do you have any suggestion please?
Hi Meric,
The problem maybe not related to the incubation time rather than the antibody concentration or the type of your antibody (polyclonal antibodies cause such a problem).
This antibody is validated for IHC in mouse tissues only. It may not be effective in westerns. I suggest finding a western-validated antibody for your species. If you want to continue using this antibody, you could try different blocking solutions, different antibody concentrations and overnight incubation at +4. Do you have a positive control to run? That would help also.
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