For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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 October 2016
Protocol Id: 10
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-4 Antibody detects endogenous levels of total caspase-4 protein. Processing intermediate forms of caspase-4 are observed at 40 kDa and 32 kDa as previously reported (7). The antibody does not cross-react with other caspases.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile125 within the p20 subunit of human caspase-4 protein. Antibodies were purified by protein A and peptide affinity chromatography.
Caspase-4 (TX/ICH-2/ICErelII) is a member of the caspase family of proteases that play a key role in the execution of apoptosis and activation of inflammatory cytokines (1-3). Expression of caspase-4 has been observed in most tissues except brain, with highest levels in placenta, lung, spleen, and peripheral blood lymphocytes (PBL). Caspase-4 was originally found to contribute to Fas-mediated apoptosis (4). Several caspases (including caspase-4, caspase-5, and mouse caspase-11 and -12) are most closely related to caspase-1 and are capable of inducing apoptosis when over-expressed but are better characterized in the proteolytic activation of inflammatory cytokines (5). Caspase-4 associates with TRAF6 and is involved in the LPS inducible production of inflammatory cytokines IL-8 and MIP1 in THP-1 cells (6). While caspase-4 and mouse caspase-12 localize to the endoplasmic reticulum (ER) and may be activated by drugs that induce ER-stress (7), at least one study suggests that caspase-4 and caspase-12 are not essential for the ER-stress induced apoptosis (8).
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|4450S||100 µl (10 western blots)||$ 255.0|