Western blot analysis of extracts from COS cells, mock transfected (-) or transfected with a construct overexpressing mouse caspase-8 (+), using Cleaved Caspase-8 (Asp387) Antibody (Mouse Specific).Learn more about how we get our images.
Western blot analysis of extracts from CTLL-2 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hours), using Cleaved Caspase-8 (Asp387) Antibody (Mouse Specific).Learn more about how we get our images.
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.
Cleaved Caspase-8 (Asp387) Antibody (Mouse Specific) detects endogenous levels of mouse and rat caspase-8 when cleaved at Asp387. This antibody will detect cleavage products containing the pro-domain with the p18 subunit (p43) as well as the p18 subunit alone.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues adjacent to the Asp387 cleavage site of rodent caspase-8. Antibodies were purified by protein A and peptide affinity chromatography.
Apoptosis induced through the CD95 receptor (Fas/APO-1) and tumor necrosis factor receptor 1 (TNFR1) activates caspase-8 and leads to the release of the caspase-8 active fragments, p18 and p10 (1-3). Activated caspase-8 cleaves and activates downstream effector caspases such as caspase-1, -3, -6, and -7. Caspase-3 ultimately elicits the morphological hallmarks of apoptosis, including DNA fragmentation and cell shrinkage.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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