Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (5 hr, 25μM), using Caspase-2 (C2) Mouse mAb.
|MW (kDa)||12, 14, 48|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 19
Caspase-2 (C2) Mouse mAb detects endogenous levels of procaspase-2 as well as its 14 and 12 kDa cleaved fragments.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal portion of human caspase-2.
Caspase-2 (Nedd2/ICH-1) is a Class I caspase with a long prodomain necessary for nuclear localization. Upon activation of the apoptotic pathway, the procaspase is cleaved at Asp316, producing a 14 kDa fragment and a 32 kDa prodomain/large subunit. Subsequent processing at Asp152 and Asp330 produces an 18 kDa large subunit and a 12 kDa small fragment (1). Caspase-2 is the nuclear apoptotic respondent to cellular genotoxic stress or mitotic catastrophe. Activation occurs upon recruitment to a complex containing a p53-induced death domain protein, PIDD (2). This suggests caspase-2 can be a nuclear initiator caspase with a requirement for caspase-9 and caspase-3 activation in downstream apoptotic events (3, 4). In apoptotic pathways resulting from UV-induced DNA damage, processing of caspase-2 occurs downstream of mitochondrial dysfunction and of caspase-9 and caspase-3 activation, extending a possible role for caspase-2 as a parallel effector caspase (5).
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