This antibody gave a positive signal in both Human and Mouse brain tissue lysate.
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pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 31 kDa (predicted molecular weight: 31 kDa).
Use a concentration of 5 µg/ml.
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.
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: OTUB1 knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: Hek293 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab101471 observed at 31 kDa. Red - loading control, ab18058, observed at 130 kDa.
ab101471 was shown to recognize OTUB1 in wild-type HAP1 cells as signal was lost at the expected MW in OTUB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1µg/mL and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Western blot - Anti-OTUB1 antibody (ab101471)
All lanes : Anti-OTUB1 antibody (ab101471) at 1 µg/ml
Lane 1 : Brain (Mouse) Tissue Lysate Lane 2 : Human brain tissue lysate - total protein (ab29466)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 31 kDa Observed band size: 31 kDa Additional bands at: 62 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab101471 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab101471, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, goat anti-rabbit DyLight® 488 (IgG H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min)HeLa, HepG2 and MCF7 cells at 5µg/ml.