western blot of phosphorylated proteins - (Mar/22/2007 )

Hi everybody

I´m trying to improve my western blots of phosphorylated proteins. I´m using fetal serum bovine (FBS) to block the membranes.
Anybody of you use bovine seroalbumin (BSA) to tblock it? Could the results be improved with BSA instead of FBS?

Thanks in advance

-mac16-

I never used FBS to block.

I always used 3%BSA in TBS (tris 20 mM NaCl 150 mM), and then incubated the antibodies in TBS-T (tween-20 0.05%) with 0.3% BSA.
I had good results.

-Missele-

QUOTE (mac16 @ Mar 22 2007, 11:33 AM)

you may use synthetic ready-to-use blocking solutions; or have a trial with skim milk solution

-The Bearer-

QUOTE (The Bearer @ Mar 22 2007, 08:24 AM)

QUOTE (mac16 @ Mar 22 2007, 11:33 AM)

you may use synthetic ready-to-use blocking solutions; or have a trial with skim milk solution

the problem with milk is that it contains phosphoproteins (eg-casein) which may lead to a high background if you are using a phospho-specific antibody.

-mdfenko-

we used to block with BSA for one particular antibody. Others 5% milk.

You have to compare blots side by side to know if it works optimally for the antibody.

-scolix-