Western blot analysis of various cell lines using DR3 (D4O3X) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS cell line source: Dr. Abraham Kapas at the University of Cambridge.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full length human DR3 (hDR3-Myc/DDK; +) using DR3 (D4O3X) Rabbit mAb.
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
DR3 (D4O3X) Rabbit mAb recognizes endogenous levels of total DR3 protein. A band of unknown origin is detected at at 110 kDa in some cell lines.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu335 of human DR3 protein. The antigen resides within the cytoplasmic domain of DR3.
The tumor necrosis factor receptor family, which includes TNF-RI, Fas, DR3, DR4, DR5, and DR6, plays an important role in the regulation of apoptosis in various physiological systems (1,2). The receptors are activated by a family of cytokines that include TNF, FasL, and TRAIL. They are characterized by a highly conserved extracellular region containing cysteine-rich repeats and a conserved intracellular region of about 80 amino acids termed the death domain (DD). The DD is important for transducing the death signal by recruiting other DD containing adaptor proteins (FADD, TRADD, RIP) to the death-inducing signaling complex (DISC), resulting in activation of caspases.
DR3/WSL-1/Apo-3/TRAMP/LARD is a TNFR family member containing the characteristic extracellular cysteine-repeats, transmembrane region, and an intracellular DD (3-7). DR3 is activated by its ligand Apo-3L/TWEAK to induce apoptosis and activation of NF-κB (8,9). Like TNF-R1, DR3 binds to the DD adaptor protein TRADD, which can then associate with other DD proteins like FADD and RIP as well as members of the TRAF family (3,4). Tissue expression of DR3 is very restricted, primarily seen on the surface of activated thymocytes and lymphocytes and plays an important role in thymocyte negative selection (3,4,10). Studies have also indicated an association with DR3 and rheumatoid arthritis (11,12).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
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