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troubleshooting westerns - (Apr/14/2005 )

I'm confused. I had western conditions for P-gp optimised. Recently i've gone back to try and do more blots, and now i'm not getting a signal at all, despite repeating the conditions identically on the same protein sampes. I've tried new batches of primary and seconday antibodies, and the detection reagent from another lab, but alas to no effect. I've also stained the blots after attempting immunostaining with the sypro stain and the protein appears to have transferred well. Any suggestions as to what subtle parameter change may account for this? Or any variation on blocking/incubation conditions to enhance antibody binding? At present i blcok over night in 10% milk and incubate with the antibodies in the presence of 5% milk.



i dont know which antibodies ur using. if ur using any phospho-specific antibody u should never use blocking buffer containing milk powder. this will cause an intense background signal as there are many serine- threonine phosphorylated proteins present in milk powder.always, use TBS buffer containing 3% or 5% BSA and 0.1% Tween 20.i am telling from experience!! for the secondary antibody u can use 10% skim milk powder in TBS containing 0.1% Tween 20.
i think even otherwise u shd. use BSA as the blocking agent.

i hope this will help u

gud luck!! smile.gif