CSF extract, sara'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


Sara's CSF EXTRACT
 
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.
   
   
Preparation:
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.
   
   
2-3 L of 1X MMR, make night before, store at 20°C.


200mL de-jelly solution, 2% cysteine, pH to 7.8 with 0.9mL 10N NaOH.
5x H1 Kinase Buffer - Amount per reaction: 2µl  Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4


750mL XB:
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
10mM K-HEPES
50mM sucrose
1X XB
11uL 10N KOH per 100mL to keep pH 7.7


250mL CSF-XB:
g32-P ATP - Amount per reaction: 0.1 µl
250mL XB (from above)
5mM EGTA
1mM MgCl2


100mL CSF-XB + PIs:
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl  Final Concentration: 1.25 µg
100mL CSF-XB (from above)
10 ug/mL LPC


2M Sucrose
H2O 7.675 µl
 
LPC
 
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. 
Cytochalasin
 
5x H1 Kinase Buffer is stored in aliquots at -20°C.
40X Energy Mix
 
Histone H1 is made up in water and stored at -20°C
Sperm
 
Extract Fix:
60% Glycerol
1X MMR
1ug/mL Hoescht
10% Formaldehyde
 
Sperm Dilution Buffer (5X)
 
10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer
   
   
If the extracts will be spun onto coverslips, then also need:
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.
 
Fixation Buffer:
BRB80
20% Glycerol
0.5% TX-100
4% Formaldehyde
 
Cushion:
BRB80
30% Glycerol
 
-20°C MeOH
   
   
 
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. 
 
Making a CSF Extract
   
   
Collect eggs.
  1.   Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
 
Wash eggs in MMR 2-3X.
 
Remove as much MMR as possible, and pour on dejellying solution.
 
De-jelly 7-10 min.  Rinse 1X in de-jelly. While eggs are de-jellying, add 1mL CSF-XB +PIs to centrifuge tubes. Add 10uL 10mg/mL cytochalasin and flick tube well.
 
Wash de-jellied eggs 3-4X in XB.
 
Wash eggs 2-3 X in CSF-XB.
 
Wash eggs 2X in CSF-XB + PIs. Leave in small volume of CSF-XB+PIs.
 
Gently pipet eggs into centrifuge tubes, put pipet below meniscus.
 
Remove excess buffer.
 
Spin 10 sec at 1500 rpm (setting 4) in clinical centrifuge/
 
Aspirate off buffer.  Add 100uL Versilube to top
 
Pack eggs: spin at 2000rpm (setting 5) for 30 sec and full speed for 1min in clinical centrifuge.
 
Aspirate off versilube and buffer from top of eggs.
 
Centrifuge packed eggs at 10,000 rpm for 15 min at 16°C in the SW55.1 rotor.
 
Collect cytoplasmic layer of extract with 18-guage needle into a 1-cc syringe.  Avoid bubbles. 
 
Transfer to new tube and estimate volume.
 
Add 1/1000 volume LPC.
 
Add 1/1000 volume cytochalasin.
 
1/40 volume 40X energy mix.
 
Add 1/40 volume 2M sucrose.

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.