Immunoprecipitation Protocol - Cell Lysates

[Luke]

Immunoprecipitation protocol

Buffers/Reagents
•   Lysis Buffer
o   20mM Tris-HCl pH 7.5
o   150mM NaCl
o   1mM EDTA
o   1mM EGTA
o   1% IGEPAL
o   2.5mM Sodium Pyrophosphate
o   1mM Sodium orthovanadate (add fresh) (can also use Phospho I cocktail)
o   1g/mL each pepstatin and leupeptin (add fresh)
o   1mM PMSF (add fresh)
o   Protease Inhibitor cocktail (add fresh)
•   3X SDS Sample Buffer
o   187.5mM Tris-HCl pH 6.8
o   6% SDS
o   30% glycerol
o   150mM DTT
o   0.03% Bromophenol Blue


1.   Lyse cells using lowest volume possible, ~500uL per 10 million cells.
2.   Pippette up and down, cells can also be sonicaed, vortexed etc.
3.   Incubate on ice
4.   Centrifuge 5 minutes to pellet cell debris
5.   Transfer supernatant to new tube (this is cell lysate)
6.   Keep 20-30uL for lysate gel, use the rest in IP.
7.   Add 15-20uL of antibody-sepharose slurry (1-5ug antibody) to lysates (alternatively primary can be incubated with lysates overnight, followed by incubation with 20uL protein A or G beads for 1-2 hours, this however leaves Ig bands on the western)
8.   Incubate with rocking or rotisserie overnight at 4 C.
9.   Centrifuge 30 seconds at 4 C, discard supernatant
10.   Wash beads 5-6 times with 500uL lysis buffer
11.   Resuspend pellet in 20-30uL SDS sample buffer
12.   Heat sample 95-100 C 5 minutes
13.   Centrifuge 30 seconds, move supernatant to new tube or load to gel
14.   Samples can be kept at -80 C