Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (ab219581)

Overview

  • Product name
    Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free
    See all SQSTM1 / p62 primary antibodies
  • Description
    Rabbit monoclonal [EPR4844] to SQSTM1 / p62 - BSA and Azide free
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human SQSTM1/ p62 aa 400 to the C-terminus. The exact sequence is proprietary.

  • Positive control
    • WB: MCF-7; HeLa; SKBR-3; 293T. mouse and rat brain, heart and lung lysates. ICC/IF: HeLa cells (untreated and treated with chloroquine), HAP1 cells (HAP1-SQSTM1 knockout cells used as negative cell line) Flow Cyt: HeLa cells.
  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®).

    ab219581 is a PBS-only buffer formulated version of ab109012, containing no BSA or sodium azide, ideal for antibody labeling. Please refer to ab109012 for information on protocols, dilutions, and image data.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab219581 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
ICC/IF Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 62 kDa.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

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

  • This WB data was generated using the same anti-SQSTM1/p62 antibody clone, EPR4844, in a different buffer formulation (cat# ab109012).

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: SQSTM1 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: HepG2 whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - unpurifed ab109012 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109012 was shown to specifically react with SQSTM1 in wild-type HAP1 cells. No band was observed when SQSTM1 knockout samples were used. Wild-type and SQSTM1 knockout samples were subjected to SDS-PAGE, Ab109012 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SQSTM1 / p62 with purified ab109012 at 1:50 dilution (10 µg/ml) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide�(ab109012)
  • Purified ab109012 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 ab109012 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

  • Overlay histogram showing HeLa cells stained with unpurified ab109012 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109012, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

  • This ICC/IF data was generated using the same anti-SQSTM1/p62 antibody clone, EPR4844, in a different buffer formulation (cat# ab109012).

    Purified ab109012 staining SQSTM1 in wild-type HAP1 cells (top panel) and SQSTM1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab109012 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).

References

This product has been referenced in:
  • Gentry EG  et al. Rho Kinase Inhibition as a Therapeutic for Progressive Supranuclear Palsy and Corticobasal Degeneration. J Neurosci 36:1316-23 (2016). Read more (PubMed: 26818518) »
  • Fiuza-Luces C  et al. Muscle Signaling in Exercise Intolerance: Insights from the McArdle Mouse Model. Med Sci Sports Exerc N/A:N/A (2016). WB . Read more (PubMed: 27031745) »
See all 5 Publications for this product

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