Immunoprecipitation protocol specific for Smac Antibody (NB500-213)

Procedure Guide for NB 500-213 Monoclonal anti-Survivin (clone 58.22)

Immunoprecipitation Procedure
1) Lyse cells plated in a 60mm dish:
a) 300 ul CHAPS buffer [50mM Tris-HCl, pH 7.5/50mM NaCl/1mM EDTA/1% NP-40/0.1% CHAPS/1mM NaVO4/1mM PMSF]
b) rock for 20 minutes at 4C
2) Harvest lysate and spin down the insoluble material at 14K rpm.
3) Collect soluble fraction.
4) Pre-clear lysate with 40 ul of 50:50 slurry of Protein A beads, rocking for 1 hour at 4C.
5) Spin down beads at 2K rpm, at 4C.
6) Collect pre-cleared lysate.
7) Incubate lysate with 5-7ug of anti-Survivin (NB 500-213) overnight, rocking at 4C.
8) Add 50 ul of Protein A 50:50 slurry for 2 hours, rocking at 4C.
9) Wash beads with 200 ul of CHAPS buffer, three times.
10) Denature immune complex by adding 2x Sample Buffer, containing 2-ME.
11) Boil for 10 minutes and load onto an SDS-gel.