Even though I use TBST 5% BSA for my blocking and phospho-antibody incubation, I get a lot of background (the film is completely black at the membrane location). I block for 2 hours at RT, primary overnight 4C, I wash 3 times 15 minutes with TBST at RT, 1 hour of secondary HRP in BSA TBST, 3 washes and ECL.
I started to worry about the secondary antibody that is normally used with milk without any background but I used BSA because of the phospo primary antibody.
Would that be OK to switch to 5% milk TBST after the first washes to make the secondary more specific or would that ruin the specificity of the primary one?
Thank you ,
Julien
Hi Julien,
it depends how strong your phospho-antibody binds to your protein of interest and if it could be sequestered by milk the phospho-proteins. You might need to increase the amount of your primary antibody.I had once the following situation using a phospho-specific antibody directed against a serine-phosphorylated protein. My colleague used to block with milk instead BSA. However, I blocked with 5% BSA TBST and could dilute the primary antibodies about 2-4-fold.
You could try using less secondary antibody to reduce the background since BSA blocks less efficiently than milk.
I hope this is helpful.
Best regards,
Christian
Thank you. I also contacted Cell Signaling tech support at the mean time and they surprisingly suggest to block with milk but incubate with the primary in BSA and then do the rest with milk (ie secondary antibody).
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