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Protocol for in vitro Kinase Assays

 

Materials needed:

  • 10X Kinase Buffer: 200mM Tris-HCl, pH 7.5, 10mM MgCl2, 250mM KCl, 10mM DTT, 400μg/mL BSA.
  • 100μM γ-32P-ATP (use 7.5x10-4 μCi per reaction, or 0.15μL of a 5mCi/mL stock per reaction)
  • 10mM ATP (use a final concentration of 100μM).
  • Substrate of choice (1μg)
  • Kinase of choice (1-2μL, depending on concentration of stock material).
  • Activators or inhibitors, if desired.
  • SDS-PAGE Sample Buffer

 

Procedure: (Keep all tubes on ice prior to reaction).

 

1. Turn on Geiger counter in radioactive area and thaw an aliquot of radioactive nucleotide (γ-32P-ATP).

 

2. Back at your bench, aliquot substrates to be tested into appropriate amount of tubes.  If the concentration of one substrate is significantly lower than another, dilute the more concentrated substrate accordingly so the volumes and concentrations are the same.  Proceed in a similar fashion for any activators or inhibitors.

 

3. Aliquot appropriate kinase to each tube (this can be done in a master mix, if possible).

 

4. Make up 1X Kinase Reaction Mix, omitting the radioactive nucleotide.  We generally use reaction volumes of 20μL.  However, all reactions can be scaled up if necessary.

 

5. Take all reaction tubes and the Kinase Reaction Mix to the radioactive area.  Add the appropriate volume of radioactive nucleotide (γ-32P-ATP) to the Kinase Reaction Mix.  Gently agitate tube to mix, and do a quick spin down to remove any material from the top of the tube.

 

6. At 10-20 second intervals, sequentially aliquot Kinase Reaction Mix in the appropriate volume (to yield 20μL reactions), to your tubes.  Allow the reactions to proceed for 10 minutes.  At 10 minutes, begin to sequentially aliquot an appropriate volume of SDS-PAGE sample buffer to each reaction to quench activity. 

 

7.  Samples may then either be frozen at -20˚C or directly run on an SDS-PAGE gel for analysis.

 

 

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia