Overview

  • Product name
    Anti-SQSTM1 / p62 antibody [EPR18351]
    See all SQSTM1 / p62 primary antibodies
  • Description
    Rabbit monoclonal [EPR18351] to SQSTM1 / p62
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, IHC-P, WB, ICC/IF, IPmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein within Human SQSTM1/ p62 aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: Q13501

  • Positive control
    • WB: Human liver and kidney lysates; HepG2 and HeLa whole cell lysates. IHC-P: Human hepatocellular carcinoma and lung cancer tissues. ICC/IF: HeLa cells (untreated and treated with chloroquine), HAP1 cells (untreated and treated with chloroquine) Flow Cyt: PC-3 cells. IP: HeLa and HepG2 whole cell lysates.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab207305 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
Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 62 kDa (predicted molecular weight: 47 kDa).
ICC/IF Use a concentration of 1 µg/ml.
IP 1/40.

Target

  • Function
    Adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. May play a role in titin/TTN downstream signaling in muscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates the interaction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels.
  • Tissue specificity
    Ubiquitously expressed.
  • Involvement in disease
    Defects in SQSTM1 are a cause of Paget disease of bone (PDB) [MIM:602080]. PDB is a metabolic bone disease affecting the axial skeleton and characterized by focal areas of increased and disorganized bone turn-over due to activated osteoclasts. Manifestations of the disease include bone pain, deformity, pathological fractures, deafness, neurological complications and increased risk of osteosarcoma. PDB is a chronic disease affecting 2 to 3% of the population above the age of 40 years.
  • Sequence similarities
    Contains 1 OPR domain.
    Contains 1 UBA domain.
    Contains 1 ZZ-type zinc finger.
  • Domain
    The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinated substrates. Mediates the interaction with TRIM55.
    The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5 and NBR1.
    The ZZ-type zinc finger mediates the interaction with RIPK1.
  • Post-translational
    modifications
    Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN.
  • Cellular localization
    Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes to the sarcomeric band (By similarity). Localizes to late endosomes. May also localize to the nucleus. Accumulates in neurofibrillary tangles and in Lewy bodies of neurons from individuals with Alzheimer and Parkinson disease respectively. Enriched in Rosenthal fibers of pilocytic astrocytoma. In liver cells, accumulates in Mallory bodies associated with alcoholic hepatitis, Wilson disease, indian childhood cirrhosis and in hyaline bodies associated with hepatocellular carcinoma.
  • Information by UniProt
  • Database links
  • Alternative names
    • A170 antibody
    • DMRV antibody
    • EBI 3 associated protein of 60 kDa antibody
    • EBI 3 associated protein p60 antibody
    • EBI3 associated protein of 60 kDa antibody
    • EBI3 associated protein p60 antibody
    • EBI3-associated protein of 60 kDa antibody
    • EBIAP antibody
    • FTDALS3 antibody
    • MGC127197 antibody
    • ORCA antibody
    • OSF-6 antibody
    • Osi antibody
    • OSIL antibody
    • Oxidative stress induced like antibody
    • p60 antibody
    • p62 antibody
    • p62B antibody
    • Paget disease of bone 3 antibody
    • PDB 3 antibody
    • PDB3 antibody
    • Phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa antibody
    • Phosphotyrosine independent ligand for the Lck SH2 domain p62 antibody
    • Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa antibody
    • PKC-zeta-interacting protein antibody
    • Protein kinase C-zeta-interacting protein antibody
    • Sequestosome 1 antibody
    • Sequestosome-1 antibody
    • SQSTM 1 antibody
    • SQSTM_HUMAN antibody
    • Sqstm1 antibody
    • STAP antibody
    • STONE14 antibody
    • Ubiquitin binding protein p62 antibody
    • Ubiquitin-binding protein p62 antibody
    • ZIP 3 antibody
    • ZIP antibody
    • ZIP3 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: SQSTM1/p62 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: HepG2 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green).

    Green - target observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab207305 and a competitor's discontinued goat polyclonal antibody.

     

     

  • ab207305 staining SQSTM1 in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (50μM, 24 hours). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab207305 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: SQSTM1/p62 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: HepG2 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab207305 was shown to specifically react with SQSTM1/p62 in wild-type HAP1 cells. No band was observed when SQSTM1/p62 knockout samples were used. Wild-type and SQSTM1/p62 knockout samples were subjected to SDS-PAGE, ab207305 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on human hepatocellular carcinoma is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • ab207305 staining SQSTM1/p62 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab207305 at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red) followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305) at 1/1000 dilution

    Lane 1 : Human liver lysate
    Lane 2 : Human kidney lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

    Predicted band size: 47 kDa
    Observed band size: 62 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).

  • All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305) at 1/5000 dilution

    Lane 1 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate
    Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 47 kDa
    Observed band size: 62 kDa why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).

  • Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and weakly nucleus staining on human lung cancer is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded human liver tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on Hhman normal liver tissue. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling SQSTM1 / p62 with ab207305 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

  • SQSTM1 / p62 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab207305 at 1/40 dilution.
    Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
    Lane 1: HeLa whole cell lysate 10ug (Input).
    Lane 2: ab207305 IP in HeLa whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HeLa whole cell lysate.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 1 second.

  • SQSTM1 / p62 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab207305 at 1/40 dilution.
    Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
    Lane 1: HepG2 whole cell lysate 10ug (Input).
    Lane 2: ab207305 IP in HepG2 whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HepG2 whole cell lysate.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 1 second.

References

This product has been referenced in:
  • Lin P  et al. An autophagy-related gene expression signature for survival prediction in multiple cohorts of hepatocellular carcinoma patients. Oncotarget 9:17368-17395 (2018). Read more (PubMed: 29707114) »
  • Chen C  et al. LncRNA HULC mediates radioresistance via autophagy in prostate cancer cells. Braz J Med Biol Res 51:e7080 (2018). Read more (PubMed: 29694502) »
See all 8 Publications for this product

Customer reviews and Q&As

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1-3 of 3 Abreviews

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (human kidney)
Total protein in input
200 µg
Immuno-precipitation step
Protein A
Specification
human kidney

Abcam user community

Verified customer

Submitted Sep 06 2018

Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (Mouse Embyronic fibroblasts)
Total protein in input
200 µg
Immuno-precipitation step
Protein A
Specification
Mouse Embyronic fibroblasts

Abcam user community

Verified customer

Submitted Aug 22 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Mouse Embyronic fibroblasts)
Permeabilization
Yes - Digitonin
Specification
Mouse Embyronic fibroblasts
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Formaldehyde

Dr. Ryan Russell

Verified customer

Submitted Aug 10 2017

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