Anti-Phospho-Ubiquitin Ser65 Recombinant Chicken Chimeric mAb (30H3K1)
Our chicken Anti-Ubiquitin (Ser65) recombinant monoclonal antibody is a chimeric antibody derived from NeuroMab clone 30H3K1. It detects human, mouse, and rat Ubiquitin (Ser65) and is purified by affinity chromatography. KO validated. Works in ELISA, ICC, IHC, WB.
![Immunofluorescent staining for detection of phospho-Ubiquitin Ser65 in WT and PINK1-KO HeLa cells using Aves Labs’ anti-phospho-Ubiquitin recombinant chicken monoclonal antibody [30H3K1] (Cat No. 78-605). Cells were treated with 20 µM carbonyl 3-chlorophenylhydrazone (CCCP) for 4 h, then fixed by adding 4% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma, X100) in PBS for 15 min. Cells were blocked with 10% goat serum in PBS for 1 h. Then, the anti-phospho-Ubiquitin Ser65 antibody (Cat No. 78-605) was added in a solution of 1% BSA in PBS for 60 min at a concentration of 1 μg/mL. Next, cells were washed two times briefly blocked again with 10% goat serum. After, secondary antibodies that were coupled to Alexa568 (orange) at a concentration of 1:1000 and Hoechst (blue) reagent in a concentration of 1:5000 were added in a solution of 1% BSA in PBS for 60 min. Finally, cells were washed and fixed again with 4% paraformaldehyde in PBS, washed two times with PBS and imaged using a Confocal microscope. Images courtesy of Wolfdieter Springer and Fabienne Fiesel, Mayo Clinic, Jacksonville, Florida.](http://www.antibodiesinc.com/cdn/shop/files/78-605-icc-1_120x120.jpg?v=1771959835)
![Western blot detection of phospho-Ubiquitin Ser65 using Aves Labs’ anti-phospho-Ubiquitin Ser65 recombinant chicken monoclonal antibody [30H3K1] (Cat No. 78-605). 4-12% Bis-Tris Gel was loaded with lysates from HEK293 cells that were mock-treated (lane 1) or treated with 30 μM carbonyl 3-chlorophenylhydrazone (CCCP) for 6 hours (lane 2). Proteins were transferred to PVDF membrane and blocked with 5% non-fat milk powder. Anti-phospho-Ubiquitin Ser65 antibody was incubated with samples at a concentration of 1 ug/mL and then detected via chemiluminescence (2 min) using a Goat Anti-Chicken H+L, HRP conjugated secondary antibody. Anti-Phospho-Ubiquitin Ser65 recognizes endogenous proteins linked to phospho-Ubiquitin Ser65 only in the CCCP treated lysates. GAPDH was used as a loading control.](http://www.antibodiesinc.com/cdn/shop/files/78-605-wb-1_120x120.jpg?v=1771959835)
![Immunofluorescent staining for detection of phospho-Ubiquitin Ser65 in WT and PINK1-KO HeLa cells using Aves Labs’ anti-phospho-Ubiquitin recombinant chicken monoclonal antibody [30H3K1] (Cat No. 78-605). Cells were treated with 20 µM carbonyl 3-chlorophenylhydrazone (CCCP) for 4 h, then fixed by adding 4% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma, X100) in PBS for 15 min. Cells were blocked with 10% goat serum in PBS for 1 h. Then, the anti-phospho-Ubiquitin Ser65 antibody (Cat No. 78-605) was added in a solution of 1% BSA in PBS for 60 min at a concentration of 1 μg/mL. Next, cells were washed two times briefly blocked again with 10% goat serum. After, secondary antibodies that were coupled to Alexa568 (orange) at a concentration of 1:1000 and Hoechst (blue) reagent in a concentration of 1:5000 were added in a solution of 1% BSA in PBS for 60 min. Finally, cells were washed and fixed again with 4% paraformaldehyde in PBS, washed two times with PBS and imaged using a Confocal microscope. Images courtesy of Wolfdieter Springer and Fabienne Fiesel, Mayo Clinic, Jacksonville, Florida.](http://www.antibodiesinc.com/cdn/shop/files/78-605-icc-1_1600x.jpg?v=1771959835)
Immunofluorescent staining for detection of phospho-Ubiquitin Ser65 in WT and PINK1-KO HeLa cells using Aves Labs’ anti-phospho-Ubiquitin recombinant chicken monoclonal antibody [30H3K1] (Cat No. 78-605). Cells were treated with 20 µM carbonyl 3-chlorophenylhydrazone (CCCP) for 4 h, then fixed by adding 4% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma, X100) in PBS for 15 min. Cells were blocked with 10% goat serum in PBS for 1 h. Then, the anti-phospho-Ubiquitin Ser65 antibody (Cat No. 78-605) was added in a solution of 1% BSA in PBS for 60 min at a concentration of 1 μg/mL. Next, cells were washed two times briefly blocked again with 10% goat serum. After, secondary antibodies that were coupled to Alexa568 (orange) at a concentration of 1:1000 and Hoechst (blue) reagent in a concentration of 1:5000 were added in a solution of 1% BSA in PBS for 60 min. Finally, cells were washed and fixed again with 4% paraformaldehyde in PBS, washed two times with PBS and imaged using a Confocal microscope. Images courtesy of Wolfdieter Springer and Fabienne Fiesel, Mayo Clinic, Jacksonville, Florida.
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Human, Mouse, Rat
ELISA, ICC, IHC, WB
Chicken
KO Validated
Trial Size
Ships: 1-2 business days
Product Details
Ubiquitin (Ser65)
Ubiquitin phosphorylation at ser65 (pS65-Ub) is implicated in mitochondrial quality control and the molecular origins of Parkinson’s Disease. The protein kinase PINK1 phosphorylates ubiquitin at the ser65 residue in response to mitochondrial stress. This acts as a powerful "on-switch" for the E3 ubiquitin ligase Parkin increasing its enzymatic activity and recruiting it to damaged mitochondria, initiating a specialized degradation process known as mitophagy. When mitochondria are depolarized or damaged, the accumulation of pS65-Ub triggers the clearance of these damaged organelles before they can release toxic reactive oxygen species (ROS) or pro-apoptotic factors. Genetic mutations in either PINK1 or Parkin that disrupt this phosphorylation event are a primary cause of autosomal recessive juvenile Parkinsonism.
Purified by affinity chromatography
1 mg/mL
Recombinant
30H3K1
IgY
ELISA, ICC, IHC, WB
Chicken
UBC, UBB
Multiple bands
Unconjugated
The exact immunogen used to generate this antibody is proprietary information
Human, Mouse, Rat
Aliquot and store at ≤ -20°C for long term storage. For short term storage, store at 2-8°C. For maximum recovery of product, centrifuge the vial prior to removing the cap.
Liquid
This recombinant antibody is a chimeric antibody created by replacing the mouse heavy and light constant regions of clone 30H3K1 with chicken IgY heavy and light constant regions. As such this antibody retains the same binding performance as the original clone 30H3K1 but can be detected using standard anti-chicken secondary antibodies allowing flexibility for multiplexing applications. This antibody is expressed recombinantly in mammalian cells and then affinity purified from the cell culture media.
This antibody was developed with support from The Michael J. Fox Foundation.
1X PBS, 0.05% Sodium Azide pH 7.4
WB: 1:1000-1:2000
IHC: 1:500-1:1000
ICC: 1:500-1:1000
IHC: 1:500-1:1000
ICC: 1:500-1:1000
Unconjugated
No cross-reactivity reported
Phosphorylated
Ser65
Each new lot of antibody is quality control tested by Western blot on mock- and CCCP-treated HeLa lysates and confirmed to stain multiple bands only in CCCP treated lysates
These antibodies are to be used as research laboratory reagents and are not for use as diagnostic or therapeutic reagents in humans.
United States
24 months from date of receipt
HMG20; Polyubiquitin-C; phospho Ser65,Ser65-phosphorylated ubiquitin; Polyubiquitin-B; pSer65-Ub; Ubiquitin
P0CG48
UniProt Summary: Exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in proteotoxic stress response and cell cycle; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling. During ubiquitination, the acceptor ubiquitin is positioned in the active site via direct interaction with the E2 ubiquitin-conjugating enzymes such as UBE2R2.
As a monoubiquitin, its C-terminal glycine is recognized as a C-degron by Cul2-RING (CRL2) E3 ubiquitin-protein ligase complexes (PubMed:39548056)
UniProt Summary: Exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in proteotoxic stress response and cell cycle; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling. During ubiquitination, the acceptor ubiquitin is positioned in the active site via direct interaction with the E2 ubiquitin-conjugating enzymes such as UBE2R2.
As a monoubiquitin, its C-terminal glycine is recognized as a C-degron by Cul2-RING (CRL2) E3 ubiquitin-protein ligase complexes (PubMed:39548056)
7316
Shipped on ice packs