Hi I am faceing one problem with my western blot experiment. I was using purified protein (size is 37 KDa). I was bloted my protein on PDVF membran. Membrane was blocked with 5% BSA (Tween-0.05%) in 1X PBS. After that blot was probed with serum antibody (which specific to my protein) as primary antibody diluted in 3% BSA (Tween-0.05%) in 1X PBS at 1:100, 1:200, 1:500 and 1:1000 dilution. Seconary antibody ( goat anti human IgG -Y- specific) were used 1:5000. washing buffer was used with PBST(T-0.1%). Noise background is very low but I am getting a band in both (Positive and Negative controls), which is specific to requaired band, Now I was optimised without primary antibody, So I relised my secondary antibody bind directly to protein. What is the possible region, which prevent the above problems.
Your's suggestions are highly appriciated.
Thank you.
As far as I can understand the secondary antibody you use binds to the purified protein (only protein in your sample). That means it may also bind to different proteins which usually suggest that it is either a very bad antibody or its concentration is high. I would test it in a western blot with crude cell lysate to see if it binds to everything. Then I would move on to lowering its concentration (1/10000) or the incubation time. You may also try blocking with NFDM.
PS: In the title you mentioned binding to membrane but in the text you say it binds to the protein. Binding to membrane demonstrates itself with high background noise something which you did not observe.
PS2: What is your negative control for this experiment? Do you have your protein in there?
PS3: Goat antibodies are generally worse than mouse/rabbit in terms of specificity
Why did you use goat anti human IgG -Y- specific antibody as your secondary antibody ? Was your first antibody generated in humans ?
Try different secondary antibodies and different dilutions (without using primary to assess secondary-derived signals). Best is to go for a secondary with minimal cross-reactivity to human serum proteins. I assume that was your problem.
I am assuming you are using a human raised antibody correct? What species is your sample? If it's a human sample then I would suggest using antibodies from other species.
The other thing to try, as mentioned before, is less secondary Ab concentration or trying other blocking buffers.
For any enzyme immune assay first standardize the assay under defined conditions. For example, amount/ concentrations of primary, secondary antibody and keeping the antigen concentration constant. Least amount of concentration or higher dilution giving the signal could be adopted for test system. As suggested by others, block with skimmed milk powder and incubate primary ab overnight at 40 C.
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