In-Vitro Kinase Assay for Jnk-1/IRS Proteins
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Transiently
transfect cell line with cDNA +FuGENE for 24 hrs.
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Starve
(12hrs.) and stimulate with appopriate ligands/reagent
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Lyse
cells, IP, and add beads
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For
JNK-1 kinase assay, spin down anti-mouse sepharose beads and wash (3x) with
lysis buffer + (2x) times kinase buffer.
Then elute kinase off beads (overnight) with flag peptide (diluted 1:100
in kinase buffer)
Kinase Buffer (in 50mL): 1.25 mL 1M Hepes
1.25 mL 1M MgCl2
25 uL 1M DTT
270 mg b-GP
500 uL 18mg/10mL NaV
Kinase Buffer (10x) 250 uL 1M
Hepes
250 uL 1M MgCl2
5 uL 1M DTT
54 mg b-GP
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Spin
down and transfer eluted kinase to an eppendorf
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Spin
down substrate (maybe still on beads) and do appropriate washes (if necessary)
·
Seperately,
make hot kinase reaction medium:
Per pointà 20 uL kinase
5 uL hot ATP
1 uL 1mM cold ATP
3 uL 10x KB
21 uL water
·
On
shaker, add 50 uL of hot rxn. medium to
each point (at staggered time intervals) and let it proceed for 30 min.. Neutralize rxn. with cold PBS, spin down,
suck off supernatant, and boil beads (in 1x loading buffer)
·
Run
SDS-page and transfer hot protein(s) to nitrocellulose
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Autoradiography