Phospho-protein detection - (Nov/26/2011 )
Has anyone done phosphorylated protein WBs before? I find that these proteins are less abundant than that of the native form and need a special lysis buffer. Bands have been pretty faint, does that mean that its less abundant?
yes they are difficult to see , you have to add phosphatase inhibitors to the lysis buffer as Phospho stop of Roche. There are some resins based in IMAC which specifically bond phosphoproteins , but you have to test yours. Actually kinase inhibitors act on the phosphorilation rate of some kinases lowering it , so the determination of inhibition rate is more difficult. Buena suerte
We don't have any problems using this lysis buffer:
<*>150 mM NaCl
<*>50 mM Tris-HCl pH 7.5
<*>1% Triton X-100
<*>1% NP-40
<*>30 mM NaF
<*>5 mM EDTA pH 8.0
<*>1 mM DTT
<*>0.1 % SDS
<*>H2O
You have to add just before using it:
<*>1 mM Na3VO4
<*>2 mM PMSF
<*>10 µg/ml aprotinin
<*>10 µg/ml leupeptin
<*>10 µg/ml pepstatin
Regards
Irene