(could be used also to CO-IP)
Protocol was modified by Guy Sovak from the original protocol that was described in:
Burnt, SA. Et al. (1998) Cell Stress & Chaperones 3: 44-56
1. Anti Hsp90, porduct #SPA-845 (Stressgen)
2. Anti mouse IgM Agarose Conjugated Ab. (Sigma #A-4540)
3. HEGD buffer (10mM HEPES, 1mM EDTA, 10% Glycerol, 5mM dithiothreitol, pH7.4).
4. HEGDMo-BSA buffer (HEGD buffer containing 20mM sodium molybdate and 2.5% BSA)
1. Prepare secondary antibody goat anti mouse IgM agarose by rotating for 1Hr at 4°C in the presence of Hsp90 Ab. Ratio Ab of to beads 3:2 (v/v).
2. Wash IgN agarose with the bound Ab twice with ice cold HEGD buffer, and once with HEGDMo-BSA buffer.
3. Centrifuge at 8000rpm for 5 minutes.
4. Resuspend the Ab-coated agarose in 0.5ml HEGDMo-BSA, (can be left in 4°C until use)
5. Mix lysate (sample) with the the Ab-coated agarose, and incubate while rotating at 4°C for 2Hr
6. Collect the complex by Centrifuge at 8000rpm for 5 minutes.
7. Wash the pellet once with HEGDMo without BSA containg 50mM NaCl, and twice with HEGDMo no BSA no NaCl.
8. Elute immunoabsorbed complexes by incubating in 2XLSB (add 10% b-ME if desired) for 20 min at 37°C.
9. Analyse by SDS.