Reagents:
Lysis/IP buffer
Volume Stock solution
1.5 ml 5 M NaCl
1 ml 1 M HEPES, PH7.2
0.5 ml 1 M NaF
40 ul 0.5 M EDTA
0.5 ml Triton X-100
Mix well
47 ml H20
Keep on ice, add the follwoing protease inhibitor before use
0.5 ml 50 mM Na3VO4 (if phosphorylation is concerned)
0.5 ml 100% Aprotonin
5 ul 10 mg/ml Leupeptine
0.5 ml 100 mM PMSF (in isopropanol)
Procedure
Spin cell extracts at 14K rpm in cold room for 10 min.
Transfer supernatants to a 1.5 new microcentrifuge tube.
Add primary antibody to final concentration of 1 ug/ml.
Shaking in cold room for 2 to 10 hours.
Spin cell extracts at 14K rpm in cold room for 10 min.
Transfer supernatants to a 1.5 new microcentrifuge tube.
Add 15 ul of Protein-A, G or A +G beads (dependent on primary AB).
Shaking in cold room for 1 to 2 hours.
Spin down the beads at 10K rpm for 10 seconds.
Suck off the supernatants (be acreful not to take away the beads).
Add 1 ml IP/Lysis buffer and mix well.
Spin down the beads at 10K rpm for 10 seconds.
Suck off the supernatants(be careful not to take away the beads).
Add 1 ml IP/Lysis buffer and mix well.
Spin down the beads at 10K rpm for 10 seconds.
Suck off the supernatants (be careful not to take away the beads).
Add 1 ml IP/Lysis buffer and mix well.
Spin down the beads at 10K rpm for 10 seconds.
Suck off most of the supernatants and leave 20 ul in the tubes.
Add 20 ul 2 X SDS loading buffer ( including 10% b-2ME).
Boil the samples for 5 minutes.
Spin at 14 rpm for 15 second.
Load 10 ul for each sample on SDS/PAGE.
Check the IP results by Western.