IP KINASE ASSAY
Complete Lysis buffer:
Stock buffer:
20 mM HEPES (pH 7.4)
10 mM KCl
1 mM EDTA
1 mM EGTA
50 mM NaCl
10% glycerol
Add Fresh on the day of the IP
1/300 mammalian PICs
50 mM NaF (0.21g/100mL)
50 mM glycerol-phosphate (1.08g/100mL)
Check pH to be sure it has not changed with addition of NaF and glycerol-phosphate
IgG Sepharose
IgG Sepharose 6 Fast Flow (Amersham biosciences 17-0969-01)
To prepare beads, place desired amount in epindorf and mark volume with marker on side of epindorf, pellet at low speed (3,000 rpm x 2 min), take of top liquid and replace with lysis buffer to original volume.
Kinase Reaction Buffer
3 uL 10X kinase buffer (lab stock)
6 uL 10 mM gamma32-ATP (5 uL hot ATP into 200 uL cold 10 mM ATP)
2 uL 10X diluted Ugp1 (substrate 1656A)
19 uL dH20
30 uL total
Method
1) grow 50 mL of the appropriate culture, pellet, and freeze at –80°C.
2) Resuspend pellets in 2 mL complete lysis buffer and lyse in 3.0 g of beads (1 min vortex x 3). Remove each liquid sample into pre-marked centrifuge tubes. To retrieve more sample from beads, add 1 mL lysis buffer to beads, vortex, and add supernate to the rest of the sample. Centrifuge at 20,000x g for 20 min to get rid of debris
3) Add half of each sample (<1.5 mL) to an epindorf with 50 uL of protein G agarose beads (washed and resuspended in lysis buffer) and rotate at 4°C for 2 hours. Half is for kinase reaction and half for western. You may also split after wash if desired.
4) Wash 4 x in lysis buffer
Kinase Reaction
Resuspend beads in 30 uL reaction mixture and run kinase reaction at 30’C for 12 min (vortex each sample twice during this time to mix). Add SDS buffer (15 uL of 4x) and heat at 98’C for 90 sec before loading on a 10 lane, 8% SDS-PAGE gel along with prestained protein standards. Run until bottom band is almost to the edge of gel (this will be free ATP). Cut off the free ATP at the end of the gel, coomassie stain, destain, and let the gel soak in water for at least 30 min. Dry gel, and expose UGP1 phosphorylation by film or by phosphorimager.
Western
Resuspend samples in 30 uL of 2x SDS sample buffer, on 8% SDS-PAGE. Transfer to PVDF membrane and then block 1 hour in 1% powdered milk PBST. Add PAP (sigma P-1291) at a 1/5000 dilution and shake gently OVERNIGHT at 4°C. Wash 3x in PBST for 15 min and room temp. Develop with picoWest (try 2 min, 4 min and 8 min time points.)
