Antibodies Plant/Algal / Environmental Stress / Heat shock

Co-Immunoprecipitaion using Agrisera HSP70 cytoplasmic antibody

2-3 µl of Agrisera anti-Hsp70 cytosolic antibody is added to ProteinA in IP buffer:
50 mM tris pH 7.5
100 mM KCl
10 mM MgCl2
rotation with antibody: 2-3 h at 4ºC

The unbound antibody must be washed in the same buffer 3-4 times

Protein extract with the concentration not lower then 3-5 mg/ml is added to ProteinA with anti-Hsp70 in amounts of 400-600 µl and is rotated for ON at 4ºC.

Unbound material is washed by IP buffer twice, then twice in the same IP buffer which contains 0.25 M NaCl.

All supernatant is carefully removed, SDS-PAGE sample buffer is added to the beads, and boiled for 10 min. After the centrifugation (12500 rpm 5 min) supernatant is loaded on a gel.

Courtesy Dr. Rena Gorovits, The Hebrew University of Jerusalem, Israel