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

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Mike's CSF Extract Protocol
Histone H1 Kinase Assay from Xenopus Extracts


1. Five days prior to planned extract date, prime frogs with PMSG, 50 units/frog (this dramatically improves the amount of extract l).


2. Approx 16hrs before preparing extract, inject frogs with Human Chorianic Ganadotropin (HCG), 600 units/frog.
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.


3. Place 4-6 liters of H2O in 20 °C incubator. 
5x H1 Kinase Buffer - Amount per reaction: 2µl  Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4


4. If you will be collecting laid eggs place the frogs in buckets containing MMR.  If only using fresh eggs leave the frogs in water overnight.  Collecting only fresh or squeezed eggs improves egg quality but severely compromises the amount of eggs that will be collected. 
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM


5. Come in to lab early, enjoy some fresh coffee and begin to set-up for making extracts.  
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


Prepare:
H2O 7.675 µl
 
· 2-3 liters of MMR: 5 mM Na-HEPES (pH 7.8), 0.1 mM EDTA, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2.  Prepare from a 10x  or 25x stock
 
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.
· 750-1000ml XB: 10 mM K-Hepes (pH 7.7), 100mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 50 mM sucrose. Maintain pH of 7.7 w/ 11 ml of 10N KOH per 100 ml.  Make with 20x XB salts, 2 M sucrose, 1 M K-Hepes (pH 7.7)
 
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
· 250 ml CSF-XB: XB + 1 mM MgCl2 + 5mM EGTA.  Take 250 ml of XB that was just made and add MgCl2 and EGTA.
 
Histone H1 is made up in water and stored at -20°C
· 100 ml CSF-XB + Protease Inhibitors:  CSF-XB + 10 mg/ml LPC. Use 100 ml of CSF-XB just made.
 
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.
· 200 ml de-jellying solution: 2 % (w/v) cysteine in 1x XB salts, pH to 7.8 with 0.9 ml of 10N NaOH.  Use 20x XB salts and cysteine.  DO NOT PREPARE CYSTEINE UNTIL IMMEDIATELY BEFORE IT IS NEEDED.
 
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.
 
6. Collect eggs in fresh MMR; good CSF and cycling extracts depends on the quality of eggs collected.  Always take care to handle eggs and extract very gently.
  1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
 
7. Remove white puffy eggs, stringy eggs, eggs that are mis-shapen and those that generally don’t look bad.  It is generally better to compromise quality for quantity.
 
8. Wash eggs 2-3 in MMR.  Pour off as much MMR as possible.
 
9. Again, remove any puffballs and/or poor quality eggs.
 
10. Add de-jellying solution to eggs, intermittently swirling gently.  De-jellying takes 5-10 minutes.  After they are dejellyed, there volume will decrease up to five fold and they will pack much tighter. 
 
11. Pour off de-jellying solution.
 
12. Wash de-jellied eggs 2-4 times with XB.
 
13. Wash eggs 2-3 times with CSF-XB.
 
14. Wash eggs 2 times with CSF-XB + PI's.
 
15. Transfer eggs to a 2.2 ml thin, clear plastic centrifuge tube containing 2 ml of LPC and 10 ml of cytochalasin B.  The size of the tube depends on the number of eggs collect.  For lower numbers of eggs, use a smaller tube. 
 
16. Aspirate excess buffer off of the eggs.
 
17. Place in a round bottom culture tube and spin in a clinical centrifuge on low for 30 seconds.
 
18. Aspirate off as much buffer as possible and add 100 - 200 ml of versalube oil.
 
19. In clinical centrifuge spin for 30 seconds on low and 60 seconds on high. 
 
20. Aspirate ALL buffer and versalube from the top of packed eggs.
 
21. Transfer the tube to a HB-4 rotor and spin at 10,000 rpm, 15 minutes, 16 °C (or the equivalent in a similar spinning bucket rotor)
 
22. Store the crushed eggs on ice.  The top yellow layer is the lipid layer and the dark bottom layer is yolk and nuclei.  The middle layer is the desired cytoplasmic extract.
 
23. After wiping the outside of the tube with a alcohol wetted kimwipe, insert an 18-gauge needle into the side of the tube (BE CAREFUL), and draw out the cytoplasmic layer.  Use a new needle for each tube.
 
24. Place the fresh extract into a clean pre-chilled tube on ice.
 
25. Estimate the volume of extract obtained and add 10mg/ml LPC, cytochalasin, 1x final concentration energy mix, and 1/40 volume of 2 M sucrose.  Add into cap and then tip a couple of times to mix. 
 
26. Test to determine if the extract is arrested in mitosis.  To do so, take 15 mL of fresh extract and to a tube that contains 1 ml of diluted sperm for experiment.  Remove 7.5 ml and add to a tube containing 1 ml of 4 mM CaCl2.  Place at room temperature for 20-30 minutes. 
 
27. Place 1 ul of extract onto a plane glass slide and overlay with 3 ml of extract fix (100 ml 10x MMR, 0.1 ml Hoechst, 300 ml 37% formaldehyde, 600 ml of 80% Glycerol).  Examine under the microscope.  The sample containing calcium should exit mitosis and have interphase looking nuclei (rounded morphology). The sample without calcium should remain in mitosis and contain condensed mitotic chromatin. 

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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