Isolation of chromatin from egg extract and histone recovery (Shechter lab) and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages

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Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0]
Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]




Link to PDF: [http://www.shechterlab.org/wp-content/uploads/2009/07/Chromatin-Isolation-from-Egg-Extract.pdf]
link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]




PROTOCOL: Sperm Chromatin Isolation / Histone Preparation
Histone H1 Kinase Assay from Xenopus Extracts


Recipes:


ELB-CIB (Egg lysis buffer – Chromatin Isolation Buffer) - 1X Egg Lysis Buffer, containing .25M Sucrose 1mM Spermidine 1mM Spermine 0.5mM EDTA
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.
5mM DTT 0.1% Triton-X 100 1X Protease Inhibitor Cocktail (Roche) 10mM Sodium Butyrate 1X Phosphatase Inhibitor Cocktails I & II (Roche)
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.


ELB - 0.25M Sucrose 2.5mM MgCl2 50mM KCl 10mM Hepes-KOH pH 7.8 1mM DTT
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


Procedures/Steps:
g32-P ATP - Amount per reaction: 0.1 µl


1. Incubate sperm in egg extract (LSS, HSS, NPE, etc), use 10-100 μl/reaction, and 2000- 10,000 sperm/ μl. Flash freeze at time points or continue with unfrozen extract. For large-scale histone preparation: use 1-2ml of LSS extract and 10,000 sperm/ μl.
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl  Final Concentration: 1.25 µg


2. Add 900 μl of 1X ELB-CIB (ice-cold); mix thoroughly and incubate on ice for 10 minutes. Underlayer carefully with 200 μl of ELB-CIB containing 0.6M sucrose.
H2O 7.675 µl
 
3. Spin in swinging bucket rotor at 4000g for 5-10 minutes at 4C. (In eppendorf, 6000RPM). There should be a visible white pellet at the bottom of the tube.
 
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. Carefully remove supernatant with pipet and respin briefly. Aspirate remaining supernatant with pipet.
 
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
5. Wash pellet 1X with 1ml ELB-CIB. (For histone prep, include 0.3M NaCl). Completely aspirate supernatant with pipet.
 
Histone H1 is made up in water and stored at -20°C
6. For gel analysis: Resuspend pellet in 1X Laemmli loading buffer. The DNA can get viscous when hot; cool before loading on SDS-PAGE gel.
 
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. For histone prep: Extract pellet in 0.4N H2SO4 for 2 hours or overnight. Precipitate extracted proteins in supernatant with 25% cold TCA for 30 minutes on ice, spin down and wash pellet with 100% cold acetone. Dissolve pellet in appropriate volume of water.
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.

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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