Anti-OTUB1 antibody (ab101471)
Key features and details
- Rabbit polyclonal to OTUB1
- Suitable for: WB, ICC/IF
- Reacts with: Mouse, Human
- Isotype: IgG
Overview
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Product name
Anti-OTUB1 antibody
See all OTUB1 primary antibodies -
Description
Rabbit polyclonal to OTUB1 -
Host species
Rabbit -
Tested applications
Suitable for: WB, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat, Dog, Pig
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Immunogen
Synthetic peptide corresponding to Human OTUB1 aa 200 to the C-terminus conjugated to keyhole limpet haemocyanin.
(Peptide available asab113298) -
Positive control
- WB: HEK-293T, HAP1 and HeLa cell lysate; Mouse brain tissue lysate. ICC/IF: HeLa cells.
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General notes
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.
Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.
We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.
In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.
We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.
Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.
Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
Applications
Our Abpromise guarantee covers the use of ab101471 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application Abreviews Notes WB Use a concentration of 1 µg/ml. Detects a band of approximately 31 kDa (predicted molecular weight: 31 kDa). ICC/IF Use a concentration of 5 µg/ml. Target
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Function
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. -
Tissue specificity
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. -
Sequence similarities
Belongs to the peptidase C65 family.
Contains 1 OTU domain. -
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. -
Cellular localization
Cytoplasm. - Information by UniProt
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Database links
- Entrez Gene: 55611 Human
- Entrez Gene: 107260 Mouse
- Entrez Gene: 100302092 Pig
- Entrez Gene: 293705 Rat
- Omim: 608337 Human
- SwissProt: Q96FW1 Human
- SwissProt: Q7TQI3 Mouse
- SwissProt: B2RYG6 Rat
see all -
Alternative names
- Deubiquitinating enzyme OTUB1 antibody
- hOTU1 antibody
- HSPC263 antibody
see all
Images
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Western blot - Anti-OTUB1 antibody (ab101471)All lanes : Anti-OTUB1 antibody (ab101471) at 1/10000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : OTUB1 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 31 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab101471 observed at 130 kDa. Red - loading control ab8245 observed at 37 kDa.
ab101471 Anti-OTUB1 antibody was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 ug/ml and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Western blot - Anti-OTUB1 antibody (ab101471)All lanes : Anti-OTUB1 antibody (ab101471) at 1 µg/ml
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : OTUB1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Hek293 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 31 kDaLanes 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.
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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.
Exposure time: 12 minutesSecondary antibody - goat anti-rabbit HRP (ab97080)
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Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody (ab101471)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.
Protocols
Datasheets and documents
References (0)
ab101471 has not yet been referenced specifically in any publications.
Images
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Western blot - Anti-OTUB1 antibody (ab101471)All lanes : Anti-OTUB1 antibody (ab101471) at 1/10000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : OTUB1 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 31 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab101471 observed at 130 kDa. Red - loading control ab8245 observed at 37 kDa.
ab101471 Anti-OTUB1 antibody was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab101471 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 ug/ml and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 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 : Wild-type HAP1 whole cell lysate
Lane 2 : OTUB1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Hek293 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 31 kDaLanes 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.
Exposure time: 12 minutesSecondary antibody - goat anti-rabbit HRP (ab97080)
-
Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody (ab101471)
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.
