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|9508||Caspase-9 (C9) Mouse mAb||
||H M R Hm Mk|
|R||Endogenous||17, 38, 40, 51||Rabbit|
Western blot analysis of extracts from PC12 cells, untreated or staurosporine-treated (3 µM), using Caspase-9 Antibody (Rat Specific). 51 kDa (full length); 40 kDa (prodomain + large fragment cleaved at Asp368); 38 kDa (prodomain + large fragment cleaved at Asp353); 17 kDa (large fragment)Learn more about how we get our images
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
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.
Caspase-9 Antibody (Rat Specific) detects endogenous levels of full length (51 kDa) and large fragments of caspase-9. The antibody does not recognize any other cleaved caspases.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding aspartic acid 353 of rat 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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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