Assaying H1 kinase in extracts (Stukenberg lab) and XB-FEAT-12564013: 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]
=''cyp2g1''=


This is the community wiki page for the gene '''''cyp2g1'''''  please feel free to add any information that is relevant to this gene that is not already captured elsewhere in Xenbase


link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
=nomenclature changes=
01/06/2015 
Human symbol has changed for genepage ID: 12564013 From cyp2g1 to CYP2G1P. Changed the symbol to cyp2g1p and added old symbol as synonym.


3/4/2022


Histone H1 Kinase Assay from Xenopus Extracts
removed 'pseudogene' from gene name, as only human gene is thought to be non-functional.


 
=summary for Mouse CYP2G1 from AGR=
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.  
Predicted to enable heme binding activity and monooxygenase activity. Predicted to be involved in epoxygenase P450 pathway and xenobiotic metabolic process. Predicted to be active in cytoplasm and intracellular membrane-bounded organelle. Is expressed in embryo; lung; and olfactory epithelium. Orthologous to human CYP2G1P (cytochrome P450 family 2 subfamily G member 1, pseudogene). [provided by Alliance of Genome Resources, Nov 2021]
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 09:51, 8 March 2022

cyp2g1

This is the community wiki page for the gene cyp2g1 please feel free to add any information that is relevant to this gene that is not already captured elsewhere in Xenbase

nomenclature changes

01/06/2015 Human symbol has changed for genepage ID: 12564013 From cyp2g1 to CYP2G1P. Changed the symbol to cyp2g1p and added old symbol as synonym.

3/4/2022

removed 'pseudogene' from gene name, as only human gene is thought to be non-functional.

summary for Mouse CYP2G1 from AGR

Predicted to enable heme binding activity and monooxygenase activity. Predicted to be involved in epoxygenase P450 pathway and xenobiotic metabolic process. Predicted to be active in cytoplasm and intracellular membrane-bounded organelle. Is expressed in embryo; lung; and olfactory epithelium. Orthologous to human CYP2G1P (cytochrome P450 family 2 subfamily G member 1, pseudogene). [provided by Alliance of Genome Resources, Nov 2021]

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