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 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

·        Autoradiography