Using TBST buffer - protein phosphorylation (Dec/12/2005 )
It has come to my attention that I must use TBS-T buffer when I'm doing Western Blot (after the transfer.... Ab dilution and washing- All with TBS-T) for detection of phosphorylated proteins. I'm using anti- Tyr + Ser/Thr phosphatases with the RIPA buffer, what is the purpose of TBST (why can't I just use PBST...)
Personaly I didnt use PBST but both TBST and PBST serve the same purpose.I use regularly TBST.
Re: phosphorlated proteins either TBST or PBST has worked fine for me in the past.
They might have meant to tell you that you shouldn't use milk as your blocker due to the kinase activity present.
Well, since one is phosphate buffered saline, and the other Tris buffered saline, and they're saying not to use the former with phosphorylated proteins, my guess is that the phosphates in PBS interfer with detecting the phosphates on the protein.
I use TBS-Tween for everything anyway; much of our work uses alkaline phosphatase conjugated secondary antibodies...