Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® TRIAD1 siRNA Ι #13719 (+), using TRIAD1 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The TRIAD Antibody confirms silencing of TRIAD1 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.
Western blot analysis of extracts from various cell lines using TRIAD1 Antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human TRIAD1 (hTRIAD1-Myc/DDK; +), using TRIAD1 Antibody.
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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.
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 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 June 2020
Protocol Id: 10
TRIAD1 Antibody recognizes endogenous levels of total TRIAD1 protein. Based upon sequence alignment, this antibody is not predicted to cross-react with HHARI/ARIH1.
Human, Mouse, Rat, Monkey
Chicken, Bovine, Dog, Pig, Horse
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human TRIAD1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The E3 ubiquitin-protein ligase ARIH2 (TRIAD1) is an Ariadne subfamily ligase involved in the polyubiquitination of proteins designated for proteasomal degradation. The TRIAD1 nuclear protein contains an amino-terminal acidic region, a pair of RING fingers, two carboxyl-terminal coiled coil domains and a novel C6HC DRIL/IBR domain located between the RING fingers. Together, the paired RING fingers and DRIL/IBR domain form a highly conserved TRIAD (two RING fingers and DRIL) domain (1). Research studies suggest that TRIAD1 mediates both Lys48 and Lys63 protein polyubiquitination and acts as a negative regulator of myelopoiesis. TRIAD1 ubiquitin ligase inhibits myeloid cell proliferation by mediating protein ubiquitination through the ubiquitin-conjugating enzymes UbcH7 and UbcH13 (2,3). Experimental deletion of TRIAD1 in mice has a lethal effect, leading to death at the embryonic stage or later due to a severe, multi-organ inflammatory response. Results indicate that TRIAD1 binds IκBβ in dendritic cells and promotes the degradation of the NF-κB inhibitor (4).
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