The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Peptide ELISA: Use at an assay dependent dilution. Antibody detection limit dilution 1/64000.
WB: Use at a concentration of 0.03 - 0.1 µg/ml. Predicted molecular weight: 31 kDa.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
Hydrolase that can remove conjugated ubiquitin from proteins and plays an important regulatory role at the level of protein turnover by preventing degradation. Regulator of T-cell anergy, a phenomenon that occurs when T-cells are rendered unresponsive to antigen rechallenge and no longer respond to their cognate antigen. Acts via its interaction with RNF128/GRAIL, a crucial inductor of CD4 T-cell anergy. Isoform 1 destabilizes RNF128, leading to prevent anergy. In contrast, isoform 2 stabilizes RNF128 and promotes anergy. Surprisingly, it regulates RNF128-mediated ubiquitination, but does not deubiquitinate polyubiquitinated RNF128. Deubiquitinates estrogen receptor alpha (ESR1). Mediates deubiquitination of 'Lys-48'-linked polyubiquitin chains, but not 'Lys-63'-linked polyubiquitin chains. Not able to cleave di-ubiquitin. Also capable of removing NEDD8 from NEDD8 conjugates, but with a nuch lower preference compared to 'Lys-48'-linked ubiquitin.
Isoform 1 is ubiquitous. Isoform 2 is expressed only in lymphoid tissues such as tonsils, lymph nodes and spleen, as well as peripheral blood mononuclear cells.
Belongs to the peptidase C65 family. Contains 1 OTU domain.
In addition to ubiquitin-binding at the Cys-91 active site, a proximal ubiquitin-binding site is also present at Cys-23 Occupancy of the active site is needed to enable tight binding to the second site. Distinct binding sites for the ubiquitins may allow to discriminate among different isopeptide linkages (i.e. 'Lys-48'-, 'Lys-63'-linked polyubiquitin) in polyubiquitin substrates and achieve linkage-specific deubiquitination.
Western blot - Anti-OTUB1 antibody (ab82154)Image courtesy of an Anonymous review
Anti-OTUB1 antibody (ab82154) reactivity with reduced HEK293 cell lysate after transient transfection (48h) of control siRNA (lane 1-3) or siRNA targeting OTUB1 (lane 4 and 5). After SDS-PAGE, membranes were blocked in 5% milk TBS + 0.1% Tween for 1h at 25°C before incubation with ab82154 (1:1000 dilution in 5% milk TBS + 0.1% Tween) for 16h at 4ºC. Blots was then incubated with an anti-Goat HRP-conjugated secondary antibody before developing with ECL. Anti-tubulin blot included as a loading control.