IP Kinase Assays (With TAP Tag in Yeast)
IP KINASE ASSAY (for yeast TAP-tagged PAS kinase)
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 day of IP:
1/300th mammalian PICS
50 mM NaF (0.21g in 100 mL)
50 mM glycerol-phosphate (1.08g in 100 mL)
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.)
TAP-tag procedure (for yeast TAP-tagged PAS kinase)
Be sure all buffers are at correct pH after adding all reagents!
Complete Lysis buffer:
Stock buffer:
20 mM HEPES (pH 7.4)
10 mM KCl
1 mM EDTA
1 mM EGTA
150 mM NaCl
10% glycerol
Add fresh on day of IP:
1/300th mammalian PICS
50 mM NaF (0.21g in 100 mL)
50 mM glycerol-phosphate (1.08g in 100 mL)
Wash buffer:
same as lysis buffer without EGTA
TEV cleavage buffer:
20 mM HEPES (pH 7.4)
10 mM KCl
150 mM NaCl
10% glycerol
0.5mM EDTA
1 mM DTT (add fresh)
Calmodulin Binding buffer;
20 mM HEPES (pH 7.4)
150 mM NaCl
1 mM Mg-acetate
`1 mM imidazole
2 mM CaCl2
10% glycerol
10 mM B-mercaptoethanol (add fresh)
Calmodulin Elution buffer;
Bicarbonate buffer (pH 7.4)
2 mM EGTA
KEEP COLD THE ENTIRE PROCEDURE (4’C)
Preparing Cell Extract
Grow up 2 L of culture, pellet and freeze at –80C. Resuspend in 10 mL lysis buffer and add 4 g of beads. Lyse by 3 min. vortex (do 3-1 min intervals and cool on ice 1 min between each one). Pellet debris by centrifuging 20K x g for 20 min.
Binding to IgG Beads
Add 10 mL yeast extract to 200 uL of IgG sepharose bead suspension in a 15 mL conical tube (PHARMACIA 17-0969-01). Be sure to remove 20% ETOH from beads and replace with lysis buffer prior to use. Rotate with IgG beads for 2 hours at 4C.
TEV Protease Cleavage
Wash 3X with 4 mL of wash buffer (by pelleting and resuspending, pellet at 2000 rpm for 2 min). Resuspend beads in 4 mL wash buffer and remove 100 uL for kinase reaction. and then pellet both the 100 uL sample, and the 4 mL sample, remove the supernatant. Keep the 100 uL sample on ice until kinase reaction. Resuspend the 4 mL sample in 10 mL TEV cleavage buffer and pellet. Remove supernatant and add 1 mL TEV cleavage buffer and approximately 100 units of TEV enzyme. Close the top of the conical and rotate 2 hours at 16C. During this 2 hour period, run kinase assays.
Pellet beads and keep supernatant (PAS kinase is now in supernatant). The solution remaining in the beads my be obtained by adding an additional 0.5 mL of the TEV cleavage buffer, then pelleting again. Add this supernatant to the 1 mL already harvested.
Binding to Calmodulin Beads
Add 3 volumes of calmodulin binding buffer (4.5 mL) and 13.5 uL of CaCl2 1M per mL of IgG eluate to titrate the EDTA coming from the TEV cleavage buffer. Transfer this to 200 uL of calmodulin beads in a 15 mL conical tube. Rotate 1 hour at 4C. Wash 3x with 8 mL calmodulin binding buffer, resuspend in 8 mL and add to column. Then elute 5 fractions of 200 uL with calmodulin elution buffer.
TAKE THE 150 uL samples to the proteomics core facility for LC-MS. Run 40 uL on SDS-PAGE to see what is present and save 10 uL to perform IP kinase assays.
------IF desired------
Protein analysis
To analyze proteins on gel, precipitate the final eluates and some of the controls with TCA and load SDS-PAGE gel.
TCA precipitation: To a 1 mL protein sample (at least 5ug/mL), add 100 uL of 100% TCA and vortex. Allow protein to precipitate 30 min on ice, spin for 5 min. at 10K x g. Remove supernatant, wash once with ethanol:ether (1:1) and resuspend in a buffered solution.
**This protocol is adapted from the Seraphin Lab at the EMBL.