Isolation of mitotic chromosomes from XTC cells (Stukenberg lab) and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages

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Purification of Mitotic Chromosomes-
Histone H1 Kinase Assay from Xenopus Extracts
Mitchison and Desai Method
 
DAY BEFORE


1. Arrest XTC cells (10-15 plates) with a final concentration of 1 µg/ml of Nocodazole (1µl per 10 ml of culture media) overnight (~16hrs).  
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.  
   
   
2. Make up 10x Swelling Buffer (1x = 5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA, pH 7.2)Dilute a total of 150 ml to 1x, keeping most at RT and about 25 ml on ice.
Background: Snap freeze in liquid nitrogen 1µl samples of the extract to be analyzedStore at -80°C until you are ready to perform the kinase assay.
   
   
3. Chill HB-4 in centrifuge to 4°C.  Put 2 x 15 ml corex tubes on ice.  Chill 7 ml dounce homogenizer and tight fitting pestle on ice.  Thaw Hoechst. Make up solutions for sucrose gradient steps (see Buffers at end).  Turn on microscope in Burke Lab.
Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.
   
   
4. Collect mitotic cells by mitotic shake / blow off.
 
   
5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4
5. Pellet cells by using Jouan CR4.12 centrifuge in 50ml tubes at ~2,000 rpm for 3 minutes.
 
   
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
6. Resuspend in 10 ml of RT Swelling buffer.  
 
g32-P ATP - Amount per reaction: 0.1 µl
7. After the pellet is resuspended, add 40 ml more of RT swelling buffer.
 
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl Final Concentration: 1.25 µg
 
H2O 7.675 µl
   
   
8. Swell at RT for ~5 minutes. 
   
   
9. Prepare lysis buffer described below and make up sucrose step gradient during swelling.  
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.
   
   
10. Pellet (same as above) and resuspend vigorously in 7 ml of ice cold lysis buffer (5 mM PIPES, 5 mM NaCl, 5 mM MgCl2, 1 mM EGTA,  pH 7.2 plus 0.1% Digitonin and LPC).  Excess digitonin is spun out (or settles out before use).
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
   
   
11. Transfer the cells to a chilled 7 ml dounce homogenizer and disrupt with 15-20 strokes using a tight pestle, resting for 2 seconds after each dounce.
Histone H1 is made up in water and stored at -20°C
12. Add Hoeschst to 2µg/ml and check by fluorescence to see that spindles have been disrupted. 
13. Transfer to a 14ml round bottom, polypro falcon tube (#352029) and spin in an HB-4 at ~900 rpm for 1 min at 4°C to pellet interphase nuclei and debris.
14. Layer the supernatant (~6 ml) onto 30/40/50/60% (2ml, 2ml, 2ml, 3ml) sucrose step gradient (made up with in swelling buffer) in a chilled 15 ml corex tube. 
15. Centrifuge at 5k for 15 minutes and 4°C in HB-4 swinging bucket rotor, brake off.
16. Collect chromosome in flocculent white mass using a pasteur pipette from the interphase between layers of sucrose. 
17. Check chromosomes again with Hoechst.  If there are a lot of interphase nuclei still present spin gently (~500 x g, 30 seconds) in a chilled benchtop centrifuge.
18. Freeze mitotic chromosome in lN2.
Buffers
Swelling Buffer (1x):
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA
Lysis Buffer:
5 mM PIPES, pH 7.2
5 mM NaCl
5 mM MgCl2
1 mM EGTA
 
LPC .1% Digitonin
   
   
Stock Hoechst- 10 mg/ml
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. 
   
   
Stock Nocodazole- 10mg/ml
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. 
   
   
Coffee- Medium Roast + Half and Half + Sugar
  1.  Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.

Revision as of 09:45, 14 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.