ABS1513-I
Anti-phospho-Ubiquitin (Ser65)
from rabbit, purified by affinity chromatography
Synonym(s):
Ubiqutin, Ser65 phosphorylated, Ub, Ser65 phosphorylated
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About This Item
UNSPSC Code:
12352203
eCl@ss:
32160702
NACRES:
NA.41
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biological source
rabbit
Quality Level
antibody form
affinity isolated antibody
antibody product type
primary antibodies
clone
polyclonal
purified by
affinity chromatography
species reactivity
human
species reactivity (predicted by homology)
mouse (based on 100% sequence homology), guinea pig (based on 100% sequence homology), rat (based on 100% sequence homology), horse (based on 100% sequence homology), sheep (based on 100% sequence homology), bovine (based on 100% sequence homology), hamster (based on 100% sequence homology), nonhuman primates (based on 100% sequence homology)
technique(s)
immunocytochemistry: suitable
western blot: suitable
UniProt accession no.
shipped in
ambient
target post-translational modification
phosphorylation (pSer65)
Gene Information
human ... UBB(7314)
General description
Ubiqutin (Ub) is initially produced as a 229-a.a. Polyubiquitin-B (UniProt P0CG47) precursor protein encoded by the UBB gene (Gene ID 7314) or a 685-a.a. Polyubiquitin-C precursor protein (UniProt P0CG48) encoded by the UBC gene (Gene ID 7316) in human. The C-termini of Polyubiquitin-B (Cys229) and Polyubiquitin-C (Val685) are post-translationally removed, followed by additional posttranslational cleavages to yield multiple copies of idential 76-amino acid ubiquitin (also known as high mobility group nonhistone protein HMG-20) molecules (three copies per Polyubiquitin-B and nine copies per Polyubiquitin-C). Covalent modifications of target proteins via different Ub Lys residues are well documented events during cellular DNA repair, ER-associated degradation (ERAD), transcription activation, lysosomal and proteasomal degradation. PINK1 (PTEN induced putative kinase 1) is a Ser/Thr kinase that specifically accumulates in depolarized mitochondria, where it acts as a positive regulator of parkin (Park2) E3 ubiquitin (Ub) ligase activity by phosphorylating Ub at Ser65 as well as the corresponding Ser residue in parkin N-terminal Ub-like (UBL) domain. Phosphorylated Ub interacts with phosphorylated parkin at its GINGO0 domain in an allosteric manner, inducing a conformation change and exposing parkin RING2 domain catalytic cysteine for full ligase activity. NMR based conformation study reveals that Ser65-phosphorylated Ub exists in two conformations in solution. The major conformation displays altered surface properties, while the second phosphoUb conformation exhibits a retracted C-terminal tail by two residues into the Ub core. Ub Ser65 phosphorylation has little effect toward E1-mediated E2 charging and mainly affects the discharging of E2 enzymes to form polyUb chains. In addition, the majority of deubiquitinases (DUBs), including USP8, USP15 and USP30, are shown to exhibit impaired activity against Lys63-linked poly-phosphoUb.
Specificity
Expected to react with a broad-range of species based on 100% sequence homology.
This polyclonal antibody detects only Ser65-phosphorylated ubiquitin (Ub) and ubiquitinated proteins (including oligoUb and polyUb) containing Ser65-phosphorylated ubiquitin. No reactivity toward Ub or ubiquitinated proteins in the absence of Ub Ser65 phosphorylation.
Immunogen
KLH-conjugated linear peptide corresponding to the ubiquitin target region sequence containing phosphorylated Ser65.
Application
Detect ubiquitin Ser65 phosphorylation using this rabbit polyclonal Anti-phospho-Ubiquitin (Ser65), Cat. No. ABS1513-I, validated for use in Immunocytochemistry and Western Blotting.
Immunocytochemistry Analysis: A 1:500 dilution from a representative lot detected CCCP (Cat. No. 215911) treatment-induced ubiquitin Ser65 phosphorylation in PINK1-expressing HeLa cells.
Western Blotting Analysis: A 1:5,000 dilution from a representative lot detected GST-PINK1-catalyzed Ser65 phosphorylation of recombinant Ubiquitin.
Immunocytochemistry Analysis: A 1:200 dilution from a representative lot detected phospho-Ubiquitin (Ser65) in PINK1 expressing HeLa cells without and with CCCP treatment. (Courtesy of Dr. Noriyuki Matsuda).
Western Blotting Analysis: A 1:5,000 dilution from a representative lot detected GST-PINK1-catalyzed Ser65 phosphorylation of recombinant Ubiquitin.
Immunocytochemistry Analysis: A 1:200 dilution from a representative lot detected phospho-Ubiquitin (Ser65) in PINK1 expressing HeLa cells without and with CCCP treatment. (Courtesy of Dr. Noriyuki Matsuda).
Quality
Evaluated by Western Blotting in PINK1-expressing HeLa cells.
Western Blotting Analysis: A 1:1,000 dilution of this antibody detected cellular ubiquitin Ser65 phosphorylation induction in 10 µg of lysate from PINK1-expressing HeLa cells upon CCCP (Cat. No. 215911) treatment.
Western Blotting Analysis: A 1:1,000 dilution of this antibody detected cellular ubiquitin Ser65 phosphorylation induction in 10 µg of lysate from PINK1-expressing HeLa cells upon CCCP (Cat. No. 215911) treatment.
Target description
~17/8 and ~45-250 kDa observed. 8.565/17.112/25.66/34.21 kDa (mono-/di/tri-/tetra-ubiquitin) calculated. Bands ~45-250 kDa represent ubiquitinated proteins containing ser65-phosphorylated Ub. Uncharacterized bands may be observed in some lysate(s).
Linkage
Replaces: ABS1513
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Storage Class Code
12 - Non Combustible Liquids
WGK
WGK 1
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