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Histone H1 Kinase Assay from Xenopus Extracts

Purpose:  Test whether the H1 kinase Cdc2-CcyclinB is still active in an extract.  This is a readout for the extracts mitotic state.  If activity is high, the extract is mitotic, if it is low, the extract has exited mitosis and is in interphase.  These are normally done in our lab as a readout for the spindle checkpoint.  An extract containing sufficient sperm and nocodazole will maintain a mitotic state after being released from CSF, by the addition of calcium.

 

Background:  Snap freeze in liquid nitrogen 1l samples of the extract to be analyzed.  Store at -80C until you are ready to perform the kinase assay.

 

Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.

 

Stock Solution

Amount per reaction

Final Concentration

5x H1 Kinase Buffer

2l

80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4

ATP (10mM Stock):

0.1 l

100 M

g32-P ATP

0.1 l

 

Histone H1 (10mg/ml Stock)

0.125 l

1.25 g

H2O

7.675 l

 

 

 

Add LPC to 10g/ml, DTT to 1mM and NP-40 Substitute to 0.1%.  I usually mix NP-40 and water first and vortex into solution. 

 

5x H1 Kinase Buffer is stored in aliquots at -20C.

 

Histone H1 is made up in water and stored at -20C

 

Keep the mixture on ice until a few minutes before beginning the experiment.  At this point place at room temperature for about 5 minutes, allowing it to warm up. 

 

Extract samples are moved from the -80C freezer into an ice bucket immediately before beginning the experiment.  Reactions are started by the addition of 9l of the H1 Kinase mixture (above).  Pippete up and down to mix the kinase assay solution and the extract, and incubate at room temperature for 30 minutes.  The reactions are stopped by the addition of 20l of 2x Laemmli Sample Buffer.  Samples are run on SDS-PAGE gels, coomassie stained, destained, dried, and placed on a phosphoimager. 


 

   1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.

 

 

 

 

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