Procedure Guide for NB 500-210 Monoclonal anti-Caspase 3
Western Protocol
1) Run a 10-*15% SDS polyacrylamide gel, loading ~20 ug of cell extract per lane.
2) Transfer the proteins to a membrane.
3) Block the membrane in PT-T20 [20 mM Tris-HCl, pH 7.4 / 150 mM NaCl/0.5% Tween 20] + 5% NFDM [non-fat dry milk], for 3 hour at room temperature (RT), shaking gently.
4) Rinse the membrane twice with PT-T20.
5) Incubate the membrane in anti-Caspase 3 [NB 500-210], diluted 1:500-1:1,000 in PT-T20 + 5% NFDM, for 60 minutes at RT.
6) Wash the membrane for 15 minutes, 3 times, in PT-T20 at RT.
7) Incubate the membrane in anti-mouse conjugated to HRP (secondary antibody), diluted in PT-T20 + 5% NFDM for 60 minutes at RT.
8) Wash the membrane for 15 minutes, 3 times, in PT-T20 at RT.
9) Develop membrane in chemiluminescent reagents, as instructed by kit-vendor.
*Positive control(s): Human kidney 293 cells
To Activate the Cell Extracts:
Bring the extract to a final concentration of 5 mM dATP.
Incubate the extracts at 37C for 15-30 minutes.
