4A6 – pronephros and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages
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Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0] | |||
link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm] | |||
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. | |||
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.