Western blot analysis of Jurkat cell extracts, untreated or etoposide-treated, and NIH/3T3 and C6 cell extracts, untreated or staurosporine-treated, using Cleaved Caspase-6 (Asp162) Antibody.
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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 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
Cleaved Caspase-6 (Asp162) Antibody detects endogenous levels of the large subunit of active caspase-6 resulting from cleavage at Asp179 in human or Asp162 in mouse and rat (18 kDa). This antibody does not recognize other cleaved caspases.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of p18, the large subunit of rat caspase-6. Antibodies are purified by protein A and peptide affinity chromatography.
Caspase-6 (Mch2) is one of the major executioner caspases functioning in cellular apoptotic processes (1,2). Upon apoptotic stimulation, initiator caspases such as caspase-9 are cleaved and activated (3). The activated upstream caspases further process downstream executioner caspases, such as caspase-3 and caspase-6, by cleaving them into large and small subunits, thereby initiating a caspase cascade leading to apoptosis (4,5). One of the major targets for caspase-6 is the membrane associated protein lamin A (6). The cleavage of this protein causes cell membrane malfunction, membrane blebbing and eventual cell death.
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