Crude interphase egg extract (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/Crude-S-Phase-Egg-Extract-LSS.pdf]
link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]




PROTOCOL: Crude S Phase Xenopus Egg Extract (“LSS”)
Histone H1 Kinase Assay from Xenopus Extracts


Recipes:


MMR - 100mM NaCl 2mM KCl 1mM MgCl2 2mM CaCl2 0.1mM EDTA 10mM Hepes-KOH pH 7.8 Sterile-filter
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.


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
Sterile-filter


Other ingredients - 5mg/ml cytochalasin B 10mg/ml cycloheximide (freshly prepared in H2O) 5mg/ml aprotinin/lepeptin 1000 Units/ml HCG hormone 100 Units/ml PMSG hormone 2% L-cysteine, pH 7.7 (adjusted with KOH)
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM


g32-P ATP - Amount per reaction: 0.1 µl


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


It is very important to move through this protocol as rapidly as possible. Prepare next step while waiting for current step etc.
H2O 7.675 µl
 
1. Inject female Xenopus laevis frogs (previously primed with 75 Units PMSG 3days to 1 week prior) with 800 Units HCG about 20 hours before you want to begin. By waiting longer, you can get more eggs, but they might be more likely to lyse. Lay into 2.5 liters of 100mM NaCl/5mM Hepes pH 7.8.
 
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.
2. Per 4 frogs: Make 1L 0.5X MMR, 1L ELB and 500mL 2% cysteine (pH 7.7).
 
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
3. Optional: for very fresh eggs, when collecting eggs, hold frog in both hands, legs pressed forward, and massage the back to get a few more eggs.
 
Histone H1 is made up in water and stored at -20°C
4. Remove stringy, necrotic, or lysed eggs; discard entire batch if more than 5% from 1 frog are bad.
 
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.
5. Decant as much liquid as possible from each batch of eggs. Pick out obvious gunk or bad eggs with Pasteur pipet. Pool all eggs together and pour off as much water as possible again.
 
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. Dejelly the pooled eggs with 4 volumes 2.2% L-cysteine hydrochloride pH 7.7, swirling frequently. If you have any cysteine left, remove some of the cysteine sup’t and replace with remaining cysteine. It is critical that eggs be completely dejellied before moving to MMR washes, as seen by disappearance of space between eggs and dissolution of jelly coats in sup’t. Time the cysteine step - dejellying typically takes 4-6 minutes.
 
  1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
7. Gently wash the eggs about 5 times with 0.5 X MMR. Move rapidly through these washes to get the eggs into ELB. Eggs should be fully resuspended during each wash and briefly stirred with the wide end of a glass Pasteur pipett. After the last wash, the white (bad) eggs will typically go to the center. Suck them up with a Pasteur pipet attached to a bulb (or you can wait to do this during the ELB washes as the density difference is increased and the bad eggs are more likely to float to the top).
 
8. Wash eggs 3X with ELB containing Cycloheximide and DTT as for MMR washes above. Take sufficient time to remove all the bad eggs; it is worth it (eggs appear more stable in ELB so you can take up to 10 minutes to remove bad eggs).
 
9. Pour into a 250 mL beaker and wash one final time with ELB. Pour off buffer.
 
10. Transfer eggs to falcon 2059 tubes (or eppendorf tubes for small number of eggs, see note later), allow to settle, remove excess buffer by aspiration, and spin for 1 min at 1100 rpm (~700 x g) in tabletop swinging bucket at room temp. If you can't fit all the eggs into 8 tubes, you can pack for 30 seconds, remove the sup’t, add more eggs, and then pack them again for 1 min. Try to get the tubes as full as possible, even if that means packing twice.
 
11. Remove excess buffer by aspiration with gel-loading tip; Pipett 0.5 μl of Aprotinin/Leupetin mix and 0.5 μl of Cytochalasin B per ml of packed eggs on top of the packed eggs (5, 5, and 2.5 μg/ml final concentrations, respectively). Add additional cycloheximide to 50 μg/ml final concentration. Don’t put the eggs on ice before the spin in step 12!
 
12. The eggs are crushed by centrifugation at 10,000 rpm for 20 minutes in a HB-6 rotor in a Sorvall centrifuge at 4C. Important: the rotor is kept at room temp until used to insure that eggs are warm when crushed. Add 2-3 mls of water to the adapters to prevent tube cracking. After this spin, keep the extract on ice.
• Alternative spin-crushing method for 2-4 frogs worth of eggs: pack eggs in eppendorf tube at 800RPM in microcentrifuge and spin-crush in cold-room microcentrifuge at 18,000 RPM for 10 minutes.
 
13. Remove extract, and don't be greedy! Put a parafilm seal on the top of the tube. Puncture the side of the tube with an 18 gauge needle about ~2-3 mms above the mitochondria layer, remove the needle, insert a fresh 18-gauge needle in the hole, remove the parafilm, and in roughly 30 s, suck out the extract until just before the yolk starts to enter the needle. Start with the needle angled about 45 degrees up, and pressed all the way to the opposite side of the tube. The opening of the needle shouldpoint upwards (you can keep track of the opening by marking the pink plastic of the needle). Watch this opposite side, and suck up extract until the yellow yolk starts to enter the needle. Then reposition and turn the needle to get as much extract as possible. Stop when the remaining cytosplasmic layer in the tube is no less than 2-3 mm deep or when you can no longer harvest without significant lipid or mitochondrial contamination. Remove the needle and gently eject extract into 50 mL conical tube. A little contamination from the top (yellow yolk) is OK, just not from the bottom (dark brown mitochondria which cause apoptosis).
• Alternative extract removal method: push yolk plug to side with spatula and pipet out with a transfer pipet (2059 tube) or blue-tip (eppendorf tube).
 
14. Clarify extract by spinning 5-30 minutes at 10,000 RPM (HB-6) or 18,000 RPM (eppendorf). A longer clarifying permits cleaner chromatin binding studies.

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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