IP with DSP

Pulse cells 20ml of 10E6 for 5h
Spin down, wash once with Na/K phosphate buffer, then lyse with 1ml of 1x lysis buffer without aprotinin and leupeptin
Add 2.5mg/ml DSP (dissolve in 10ul DMSO) to lysate. Sit on ice 7min.
Reaction was quenched by adding 200ul of 1M Tris-HCl (pH7.4). Also add leupeptin and aprotinin. Ice 30min.
Spin 10min
Transfer supernatant to new eppie tubes. Add 25ul anti-myc antibody conjugated agarose. Rock 3h to O/N 4C
Wash immunoprecipitates with 1ml wash buffer (20mM MOPS pH7.0, 150mM NaCl, 0.5% NP-40, 10% glycerol). Spin 2-3K 1min
Load beads onto mini-spin column. Spin 2-3k 1min
Elute proteins 3 times with 40ul elution buffer (4%SDS, 0.1M glycin pH2.5), 2-3K 1min.
Ad 10ul of 1M DTT and 20ul of 1M Tris-HCl (pH8.0) to total 120ul of elutant. Boil 10min.
Load 15ul of product to 10% gel for western and silver staining.
The rest of product wer concentrated by MeOH/CHCl3.

About 150ul of protein sample
Add 600ul of MeOH, 150ul CHCl3, 450ul H2)
Vortex and centrifuge 13k for 10min
Discard the upper aqueous phase. Protein is in interphase
Add 600ul MeOH
Vortex and centrifuge at 13k 10min
Discard supernatant
Dry pellet.

2x lysis buffer
1% NP40
300mM NaCl
40mM MOPS pH7.0
20% glycerol
2mM Na3VO4
2ug/ml Leupeptin
5ug/ml Aprotinin