VEGFA - vegfa - somites, lateral plate mesoderm, head, hypochord and Assaying H1 kinase in extracts (Stukenberg lab): Difference between pages

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'''Antigen:''' human VEGFA
Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
 
'''Xenbase Gene Page:''' [http://www.xenbase.org/gene/showgene.do?method=display&geneId=485743 vegfa]
 
link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
'''Epitope:''' amino acids 1-140
 
 
'''Host Species/Isotype:''' rabbit polyclonal
Histone H1 Kinase Assay from Xenopus Extracts
 
'''Antigen Species:''' human
 
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.
'''Applications:''' tested for WB
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.
'''Antibody Name/Catalog Number:''' [http://www.scbt.com/datasheet-507-vegf-147-antibody.html VEGF (147): sc-507]
Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.
'''Supplier:''' Santa Cruz
 
'''Comments:''' tested for WB but not working
5x H1 Kinase Buffer - Amount per reaction: 2µl  Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4
 
'''Contributor:''' Aldo Ciau-Uitz
ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
 
'''Additional Information:''' reacts with an unknown protein
g32-P ATP - Amount per reaction: 0.1 µl
 
'''Reference(s):'''
Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl  Final Concentration: 1.25 µg
 
H2O 7.675 µl
applications abbreviations:
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. 
WB - Western blotting,
5x H1 Kinase Buffer is stored in aliquots at -20°C.
IP - immunoprecipitation,
Histone H1 is made up in water and stored at -20°C
IF - immunofluorescence,
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. 
IHC - immunohistochemistry,
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. 
EMSA - electromobility shift assay,
  1.  Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.
ChIP - chromatin immunoprecipitation,
FCM - flow cytometry

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.