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IN-VITRO KINASE ASSAY

In-Vitro Kinase Assay for Jnk-1/IRS Proteins

 

        Transiently transfect cell line with cDNA +FuGENE for 24 hrs.

        Starve (12hrs.) and stimulate with appopriate ligands/reagent

        Lyse cells, IP, and add beads

        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

 

        Spin down and transfer eluted kinase to an eppendorf

        Spin down substrate (maybe still on beads) and do appropriate washes (if necessary)

        Seperately, make hot kinase reaction medium:

 

Per point 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

        Autoradiography