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:
- 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.
- 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.
- 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.