|H M R Mk||Endogenous||31||Rabbit IgG|
Western blot analysis of extracts from various cell lines using OTUB1 (D8F7) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with Myc/DDK-tagged cDNA expression constructs encoding full-length human OTUB1, isoform1 (hOTUB1-Myc/DDK, +) and full-length human OTUB2 (hOTUB2-Myc/DDK, +), using OTUB1 (D8F7) Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
OTUB1 (D8F7) Rabbit mAb recognizes endogenous levels of total OTUB1 protein. This antibody does not cross-react with OTUB2 and based upon sequence alignment, is not predicted to cross-react with OTUB1, isoform 2 (ARF-1).
Human, Mouse, Rat, Monkey
Hamster, Bovine, Dog
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile82 of human OTUB1 protein.
Protein ubiquitination and deubiquitination are reversible processes catalyzed by ubiquitinating enzymes (UBEs) and deubiquitinating enzymes (DUBs) (1,2). DUBs are categorized into 5 subfamilies: USP, UCH, OTU, MJD, and JAMM (1,2). The OTU subfamily comprises a group of approximately 100 putative cysteine proteases that are homologous to the ovarian tumor gene product of Drosophila (3). OTUB1 and OTUB2 (OTU domain-containing Ubal-binding proteins) display no significant similarity to any known DUB, but are close homologs and possess an OTU domain that contains conserved cysteine, histidine, and aspartate residues that define the putative catalytic triad of cysteine proteases. Furthermore, sequence analysis of OTUB1 and OTUB2 reveals the presence of putative Ub-interaction motifs (UIMs) and Ub-associated domains (UBAs), which are characteristic of proteins that regulate protein ubiquitination. OTUB1 and OTUB2 also possess a putative nuclear localization signal (NLS) and a consensus LxxLL motif, which mediates the interaction between transcriptional co-activators and nuclear hormone receptors (4).
OTUB1 exists as two isoforms that are generated by alternative splicing; the shorter 31 kDa isoform is ubiquitously expressed, while the longer 35 kDa isoform (ARF-1) has a more restricted expression pattern and is mostly detected in lymphoid organs (5). Biochemical analysis has demonstrated that OTUB1 has a preference for cleaving K48-linked polyubiquitin chains over K63-linked polyubiquitin chains and is capable of cleaving NEDD8, but not SUMO-1, -2, and -3 or ISG15 conjugates (6). OTUB1 isoforms have been implicated in anergy induction in CD4+ T cells by regulating the stability of the E3 ligase GRAIL (5). More recently, OTUB1 was found to bind to and inhibit the E2 activity of UBE2N through a novel mechanism not involving OTUB1 DUB activity, thus compromising the ability of the E3 ligase RNF168 to drive ubiquitination-dependent repair of DNA double-strand lesions (7). OTUB1 also appears to suppress MDM2-dependent ubiquitination of p53 independent of its catalytic activity, primarily by suppressing the activity of the MDM2 cognate E2 UbcH5 (8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Anti-FLAG is a registered trademark of Sigma-Aldrich Biotechnology.
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