Cycling extracts, mike's protocol (Stukenberg lab) and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages

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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.
   
   
Mike’s Cycling Xenopus Extracts
Background:  Snap freeze in liquid nitrogen 1µl samples of the extract to be analyzed.  Store at -80°C 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.
   
   
1. Prepare extracts as is done normally. (Murray, 1991;Desai et al., 1999;Chen and Murray, 1997)  Do not centrifuge faster than 10K for 10 min at 16 deg C in HB-4 rotor (or equivalent in mini-ultra).  Higher speed spins will not cycle as well (or so I have read).   
 
5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4
 
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
 
g32-P ATP - Amount per reaction: 0.1 µl
 
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl Final Concentration: 1.25 µg
 
H2O 7.675 µl  
   
   
2. Supplement the extract with cytochalasin D, 1 X energy mix, 50 mM Sucrose and LPC.  The use of cytochalasin seems to help extracts cycle.  Although, since it is more expensive, I would use it for cycling extracts only, and not for normal CSF.
   
   
3. Bring aliquots of extract to room temperature for 1-2 minutes.  Add sperm at a concentration of  less than 1,000 sperm/ul (this can be varied, but I have read to high can overwhelm the extract) and gently flick the tube to thoroughly mix them the sperm in the extract.  Good mixing is essential, but the extract is sensitive, so be careful.   
Add LPC to 10µg/ml, DTT to 1mM and NP-40 Substitute to 0.1%.  I usually mix NP-40 and water first and vortex into solution.   
   
   
4. Add 1/10 volume of cycling solution which contains 4 mM CaCl2, 100 mM KCl, 1 mM MgCl2. Again gently flick the tube to mix thoroughly. 
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
   
   
5. At about 30-45 minutes the extracts will have exited morphologically.  This can be checked by squashing 1 µl under 3 µl of squash fix (MMR + ~10% formaldehyde + ~50% Glycerol + Hoechst).  The chromatin will appear rounded and diffuse and they should be uniform throughout the extract.  That is, all nuclei should be at almost the same point. 
Histone H1 is made up in water and stored at -20°C
   
   
6. Check the extract every 10-15 minutes on the microscope.   
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.   
   
   
7. When the chromatin is again condensed (around 90-120 minutes) add back an equal volume of room temperature CSF extract to re-arrest the cycling extract in mitosis.  After -15-20 minutes check to be sure that the chromatin is condensed in a in a tight mass.   
Extract samples are moved from the -80°C freezer into an ice bucket immediately before beginning the experiment. Reactions are started by the addition of 9µl of the H1 Kinase mixture (above).  Pipette 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 20µl of 2x Laemmli Sample Buffer.  Samples are run on SDS-PAGE gels, coomassie stained, destained, dried, and placed on a phosphoimager.   
   
   
8. Add nocodazole (if required for experiment) to a final concentration of 10 µg/ml (dilute nocodazole in extract).
  1.  Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
    
9. Continue to incubate at room temperature for 15 minutes.
References
      1.    Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
      2.    Desai, A., A.Murray, T.J.Mitchison, and C.E.Walczak. 1999. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. Methods Cell Biol. 61:385-412.
      3.    Murray, A.W. 1991. Cell cycle extracts. Methods Cell Biol. 36:581-605.

Latest revision as of 13:53, 15 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


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 1µl samples of the extract to be analyzed. Store at -80°C 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.


5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4

ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM

g32-P ATP - Amount per reaction: 0.1 µl

Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl Final Concentration: 1.25 µg

H2O 7.675 µl


Add LPC to 10µg/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 -20°C.

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

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 -80°C freezer into an ice bucket immediately before beginning the experiment. Reactions are started by the addition of 9µl of the H1 Kinase mixture (above). Pipette 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 20µl 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.
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