Greetings everyone. I’m trying to pull down a protein using immunoprecipitation. I am using protein A/G coated magnetic beads which I have successfully cross-linked with Antibody against the protein of my interest. Now I want to pull down that protein in the nuclear lysate.
After 4 hours of pull-down, I directly run the beads in SDS PAGE gel and after running blot it to PVDF membrane at 15V for 50 minutes. After transferring I block it with 5% skimmed milk in TBS buffer pH 7.5. I then keep my blot to bind primary antibody (1:750 dilution) overnight followed by 3 washes of TBS substituted with 0.1% TWEEN20. I then incubate my blot with HRP conjugated secondary antibody (1:10,000 dilution) and wash my blot with TBS substituted with 0.1% TWEEN-20 thrice. I then add HRP substrate over the blot and read chemiluminescence. That's when very huge blotches and I think my band of interest gets hidden in the blotches, even after minimal exposure I get these black patches.
I even tried increasing TWEEN 20 percentage as well as increasing the number of washing as well as washing time but to no success.
I have attached the image of the blot.
Any suggestions will be highly appreciated. Thanks in advance.
Are you presoaking the PVDF membrane in methanol or ethanol before blotting?
Adam B Shapiro yes, I do wash my membrane with methanol for 1 minute before blotting.
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