Western blot analysis of extracts from NIH/3T3 cells, untreated, staurosporine-treated (1 µM), or cytochrome c-treated (0.25 mg/ml), using Caspase-9 Antibody. p39: caspase-9 cleaved at Asp368. p37: caspase-9 cleaved at Asp353.
|MW (kDa)||37, 39, 49|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Caspase-9 Antibody (Mouse Specific) detects endogenous levels of both full length mouse caspase-9 (49 kDa) and the large fragment of mouse caspase-9 resulting from cleavage at aspartic acid 353 (37 kDa) and/or aspartic acid 368 (39 kDa). The antibody does not cross-react other caspases.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the cleavage site of mouse caspase-9. Antibodies are purified by protein A and peptide affinity chromatography.
Caspase-9 (ICE-LAP6, Mch6) is an important member of the cysteine aspartic acid protease (caspase) family (1,2). Upon apoptotic stimulation, cytochrome c released from mitochondria associates with the 47 kDa procaspase-9/Apaf-1. Apaf-1 mediated activation of caspase-9 involves intrinsic proteolytic processing resulting in cleavage at Asp315 and producing a p35 subunit. Another cleavage occurs at Asp330 producing a p37 subunit that can serve to amplify the apoptotic response (3-6). Cleaved caspase-9 further processes other caspase members, including caspase-3 and caspase-7, to initiate a caspase cascade, which leads to apoptosis (7-10).
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