Western blot analysis of extracts from various cell types using E2-25K/Hip2 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
E2-25K/Hip2 Antibody detects endogenous levels of total E2-25K/Hip2 protein.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg11 of human E2-25K/Hip2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Protein ubiquitination requires the concerted action of the E1, E2, and E3 ubiquitin-conjugating enzymes. Ubiquitin is first activated through ATP-dependent formation of a thiol ester with ubiquitin-activating enzyme E1. The activated ubiquitin is then transferred to a thiol group of ubiquitin-carrier enzyme E2. The final step is the transfer of ubiquitin from E2 to an ε-amino group of the target protein lysine residue, which is mediated by ubiquitin-ligase enzyme E3 (1).
E2-25K (Hip2) is a member of the E2 protein family that catalyzes multiubiquitin chain synthesis via Lys48 of ubiquitin (2). E2-25K is reportedly involved in Alzheimer's disease, Huntington's disease and antigen processing through its interaction with amyloid-β, huntingtin, and MHC-heavy chain proteins (3-5). Lys14 of E2-25K can be modified by SUMOylation, with this modification resulting in inhibited E2 activity (6).
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