Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or treated with MG-132 #2194 (10 μM, 90 min; +), using
Ubiquitin (E4I2J) Rabbit mAb (Biotinylated) (upper) and β-Actin (13E5) Rabbit mAb #4970 (lower).
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This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Ubiquitin (E4I2J) Rabbit mAb #43124.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/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.
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 266
Ubiquitin (E4I2J) Rabbit mAb (Biotinylated) recognizes endogenous levels of free ubiquitin and polyubiquitinated proteins. This antibody is able to detect free ubiquitin, linear polyubiquitin (M1-linked), and homotypic polyubiquitin chains consisting of K6, K11, K27, K29, K33, K48, and K63 linkages.
Human, Mouse, Rat
D. melanogaster, Xenopus, Bovine, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly35 of human ubiquitin protein.
Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25A, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7).
Explore pathways + proteins related to this product.
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