For PKA phosphorylation, the medium contains 20 ug/ml (at minimum) of the peptide/protein to be phosphorylated in the presence of 100 mM KCl, 1 mM EGTA, 10 mM MgCl2, 20 mM Hepes (pH 7.4), 1 mM DTT, 0.1 mM db-cAMP (PKA activator), and 660 nM chelerythrineCl (PKC inhibitor)...In the case of membrane proteins a preincubation with 0.05% TRITON must be required, some samples must be treated with  200 nM H-89 (PKA inhibitor) to confirm PKA dependent phosphorylation....

The phosphorylation can be performed  with a pre-incubation during 20min with PKA phosphorylation medium and can be started by the addition of 0.5 mM [gamma-32Pi]ATP (sp. activity of 800,000 cpm/nmol) and stopped by addition of electrophoresis sample buffer after 1 h if you are chasing for endogenous protein kinases of after 5-10 min for assays with commercial PKC or PKA (20 mUnits of each must be enough).

SDS–PAGE analysis of the phosphorylated proteins must be done according to [Laemmli UK, 1970], with a 4% stacking gel and 15% resolving gel to perform an autoradiogram of the labeled proteins or alternatively you can reveal the results using a cyclone or phosphorimage apparatus.... Hope it will be useful to you.. Best wishes and Bye Bye...

 Many thanks Prof. Carlos for a detailed method. I have read that PKA is cAMP dependent so do i need to add cAMP at some stage? As i am new to this field therefore i find it intriguing!

Hello Arslan. Are you planning to digest with trypsin before or after you incubate with PKA? If before, how will you remove the trypsin?

Hi Bruce, 

I am planning to incubate tryptic peptides with PKA. I think by using 10kDa filter i can remove trypsin!

We use db-cAMP in place of cAMP.

10 kDa nominal MWt cutoff will remove most trypsin ... but also any larger tryptic peptides. Are you sure that all of the peptides you are interested in will pass through this filter? Any time I have used this approach, I have performed a phosphorylation reaction using [g32P]ATP and conditions appropriate for the kinase(s), resolved the protein on SDS-PAGE, dried the gel and exposed to film, excised a gel chip containing the phosphorylated protein, exposed the gel to film again to make verify the correct region of the gel was excised, and then digested the protein out of the chip using sequencing-grade trypsin dissolved in 50 mM ammonium bicarbonate buffer.

Hi Aslan..If you employ db-AMPc you are free to go...Because this is a cell permeable analog of AMPc widely chosen in signalling studies. Regardin trypsin, I think you can easily get rid of it by using a centricon or amicon with a 15-20 kDa cut-off...I think that this mol weight range is better, because you can retain a more significant number of tryptic fragments than a 10 kDa one which you surely will remove trysin but also will lose larger trytic fragments as stated by our colleague Bruce Allen...

Thanks Bruce and Carlos!