Hello,

I am desperate to see endogenous expression levels of pS6K in S2R+.

I am using Cell Signalling ab which recognizes the Drosophila protein, according to manifacture's recommendation

I saw it once and never again following 30' stimulation with 100ug EGF- I see nothing in unstimulated cells.

What I do is:

Lyse cells from 6 well plate with 100ul NP40 Lysis buffer

Load 40ul of it per well

Block in 5% skimmed milk

ab in 50% PBS and 50% PBS with 5% milk for 1h at RT

ab 1:1000 O/N

Any suggestion of what I do wrong or how to improve signal?

Thanks a bunch

Marghe