Human Phospho-Ubiquitin (S65) Antibody

  
  • Specificity
    This antibody detects mono- and poly-Ubiquitin chains that are phosphorylated on Serine 65. It has no cross-reactivity with non-phosphorylated Ubiquitin, Ubiquitin phosphorylated at Serine 57 or Tyrosine 59, or phosphorylated Parkin. Detects only recombinant, phosporylated Ubiquitin. Not recommended on natural lysates (see western blot results below).
  • Source
    Polyclonal Rabbit IgG
  • Purification
    Antigen Affinity-purified
  • Immunogen
    Peptide sequence from Ubiquitin, phosphorylated at Serine 65.
    Accession # P0CG47
  • Formulation
    0.5 mg/ml in PBS pH 7.4, 50% (v/v) Glycerol
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    0.5-1 µg/mL
    See below
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
Detection of Phospho-Ubiquitin (S65) by Western blot. Tetraubiquitin chains of each indicated linkage type were incubated for 0-4 hours in reactions containing recombinant PINK1 kinase (Cat# AP-180) and ATP. At indicated times a portion of each reaction was removed and terminated with SDS-PAGE sample buffer. SDS-PAGE gels (10-20%) were used to resolve approximately 150 ng of Ubiquitin tetramer from each reaction. Western Blots were developed using either alpha -phospho-Ubiquitin, pS65 (upper panels) or anti-Ubiquitin (Cat# MAB701, lower panel). Primary antibodies were used at 1 μg/ml in PBST + 0.5% BSA, while HRP-labeled secondary antibodies ( alpha -rabbit for A-110, alpha -mouse for MAB701) were used at a 1:10,000 dilution in PBST + 0.5% BSA. Further details are available upon request.
Detection of Phospho-Ubiquitin (S65) by Western blot. Tetraubiquitin chains of each indicated linkage type were incubated for 0-4 hours in reactions containing recombinant PINK1 kinase (Cat# AP-180) and ATP. At indicated times a portion of each reaction was removed and terminated with SDS-PAGE sample buffer. SDS-PAGE gels (10-20%) were used to resolve approximately 150 ng of Ubiquitin tetramer from each reaction. Western Blots were developed using either alpha -phospho-Ubiquitin, pS65 (upper panels) or anti-Ubiquitin (Cat# MAB701, lower panel). Primary antibodies were used at 1 μg/ml in PBST + 0.5% BSA, while HRP-labeled secondary antibodies ( alpha -rabbit for A-110, alpha -mouse for MAB701) were used at a 1:10,000 dilution in PBST + 0.5% BSA. Further details are available upon request.
Preparation and Storage
  • Shipping
    The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
  • Stability & Storage
    • 6 months from date of receipt, -20 °C as supplied.
    • 3 months, -20 °C under sterile conditions after opening.
Background: Ubiquitin
Serine/Threonine kinase PINK1 (PTEN-induced putative kinase protein 1) plays a critical role in preventing mitochondrial dysfunction during cellular stress. PINK is translated in the cytosol, then translocated to the outer mitochondrial membrane where it is rapidly cleaved and degraded as a part of normal mitochondrial function. In damaged (depolarized) mitochondria PINK becomes stabilized and accumulates, resulting in the subsequent phosphorylation of numerous proteins on the mitochondrial surface including Mfn2. Ultimately PARK2 (E3 Ubiquitin Ligase Parkin) is recruited to the damaged mitochondria where it is activated by PINK-mediated phosphorylation of PARK2 at serine 65, and PARK2 interaction with phosphorylated Ubiquitin (also phosphorylated by PINK on serine 65). This signaling cascade is critical for clearing the damaged mitochondria via selective autophagy (mitophagy) by mediating activation and translocation of PARK2.
  • Entrez Gene IDs:
    7314 (Human); 298693 (Rat)
  • Alternate Names:
    RPS27A; UBA52; UBB ubiquitin B; UBB; UBC; Ubiquitin
Related Research Areas
Isotype Controls
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Normal Rabbit IgG Control

Ctrl AB-105-C 43  
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Normal Rabbit IgG Control

Ctrl, CyTOF-ready MAB1050  
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Secondary Antibodies
Description Application Cat# Citations Images  

Rabbit IgG HRP-conjugated Antibody

WB HAF008 23  
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Donkey Anti-Rabbit IgG NorthernLights™ NL557-conjugated Antibody

Flow, IHC, ICC NL004 10
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Donkey Anti-Rabbit IgG NorthernLights™ NL637-conjugated Antibody

Flow, IHC, ICC NL005 2
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Rabbit IgG APC-conjugated Antibody

Flow F0111 1  
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Rabbit IgG PE-conjugated Antibody

Flow F0110 2  
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Rabbit IgG Fluorescein-conjugated Antibody

Flow F0112 5  
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Donkey Anti-Rabbit IgG NorthernLights™ NL493-conjugated Antibody

Flow, IHC, ICC NL006 4
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Goat Anti-Rabbit IgG Biotinylated Antibody

WB BAF008 4
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Mouse/Rabbit IgG VisUCyte HRP Polymer Antibody

IHC VC002  
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Rabbit IgG Antibody

WB AF008 1
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Rabbit IgG VisUCyte HRP Polymer Antibody

IHC VC003  
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Donkey Anti-Rabbit IgG (H+L) Antibody

D-301-C-ABS2
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