Western blot analysis of extracts from HeLa, THP-1, and YB2/0 cell lines using Caspase-8 (D35G2) Rabbit mAb.
Western blot analysis of extracts from control HeLa cells (lane 1) or Caspase-8 knockout HeLa cells (lane 2) using Caspase-8 (D35G2) Rabbit mAb #4790 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Caspase-8-knockout HeLa cells confirms specificity of the antibody for Caspase-8.
Western blot analysis of extracts from CTLL-2 cells, untreated (-) or treated with Cycloheximide #2112 (CHX, 10 μg/ml, overnight) followed by Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/ml, 4 hr; +), using Caspase-8 (D35G2) Rabbit mAb.
Western blot analysis of 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Caspase-8 protein (hCasp8-Myc/DDK; +), full-length mouse Caspase-8 protein (mCasp8; +), or full-length human Caspase-10 protein (hCasp10; +), using Caspase-8 (D35G2) Rabbit mAb.
|REACTIVITY||H M R|
|MW (kDa)||10, 57|
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 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
Caspase-8 (D35G2) Rabbit mAb detects endogenous levels of total caspase-8, including the p10 subunit of the activated protein. Caspase-8 (D35G2) Rabbit mAb may also cross-react with overexpressed levels of caspase-10.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxyl terminus of human caspase-8 protein.
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.
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