P25CDK Phosphorylation of Peptide Substrates Using Recombinant Proteins

Combine 4J.Lg ofGST-Cdk5 with 4J.Lg GST-p20 or GST-p25.

Bring total volume to ll.7J.Ll with PBS

Keep on ice until ready to add “hot substrate solution.”

Hot substrate solutionStock solutions

Ammmt per rxn

Final cone. in 30μl kinase rxn

1M MOPS, pH7.2

0.6μl

20mM

lMMgCh

O.l5μl

5mM

lMDTT

0.03μl

1mM

15mM Hlpeptide

1μl

500μM

lmMATP

0.6μl

20μM

5 J.LCi/ul y-ATP

0.5μl

ddH2-0

15.42μl

Total volume

18.3μl

 

Add 18.3J.Ll of”hot substrate solution” to the ll.7J.Ll solution  containing p25/Cdk5

Let kinase rxn proceed for lhr-5hrs at RT Add 100 J.Lg BSA (10 J.Ll of lOmg/ml)

Stop reaction with 30 J.Ll cold 20% TCA Ice 5minutes

Spin 14K for 5 minutes to precipitate proteins

Spot 28uL of supernatant on P-cellulose discs

Also spot 10 J.Ll of the “hot substrate solution” on the P-dics to obtain background counts

(usually this number is 300 or lower)

Wash discs with 4X lOmin with 0.3% Phosphoric acid (lOmls per disc)

Count in lOml safescint fluid using program 12 in Howley lab scintillation counter

Some things to keep in mind:

  1. Dissolve the peptide you want to use as a substrate in 20mM MOPS pH 7.2, 5mM MgCh (no ATP) to make a stock solution of 15mM, and keep this at -20°C.
  2. Do not use GST-p25 or GST-Cdk5 that has undergone even one freeze-thaw. The kinase activity of the complex can be seriously impaired by this process.
  3. If you are testing a new peptide as a substrate, consider doing at least one set of reactions for longer than an hour at RT.