Overview

  • Product nameAnti-SQSTM1 / p62 antibody
    See all SQSTM1 / p62 primary antibodies
  • Description
    Mouse monoclonal to SQSTM1 / p62
  • Tested applicationsSuitable for: IHC-P, WB, ICC/IF, Flow Cyt, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Rhesus monkey, Chinese Hamster
  • Immunogen

    Recombinant full length protein, corresponding to amino acids 1-441 of Human SQSTM1/ p62

Properties

Applications

Our Abpromise guarantee covers the use of ab56416 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
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB Use a concentration of 1 - 5 µg/ml.
ICC/IF Use a concentration of 10 µg/ml. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS). Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.
Flow Cyt Use 0.5µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IHC-Fr Use at an assay dependent concentration. PubMed: 22577215

Target

  • FunctionAdapter 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 specificityUbiquitously expressed.
  • Involvement in diseaseDefects 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 similaritiesContains 1 OPR domain.
    Contains 1 UBA domain.
    Contains 1 ZZ-type zinc finger.
  • DomainThe 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 localizationCytoplasm. 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
    • 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

Anti-SQSTM1 / p62 antibody images

  • Western blot against tagged recombinant protein immunogen using ab56416 SQSTM1 / p62 antibody at 1ug/ml. Predicted band size of immunogen is 75 kDa
  • Monoclonal antibody to SQSTM1 (ab56416) on HeLa cell, antibody concentration 10 ug/ml.
  • ab56416 staining SQSTM1/p62 in Human A431 epidermoid cancer cells by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with formaldehyde, permeabilized with Triton X-100 and blocked with 5% BSA for 30 minutes at room temperature.  Samples were incubated with  primary antibody (1/50) in 5% BSA for 1 hour. An Alexa Fluor® 488-conjugated Goat monoclonal to mouse IgG (1/50) was used as secondary antibody. 

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab56416 (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 (ab56416, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • ab56416 (1µg/ml) staining SQSTM1 in human lymph node using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and cytoplasmic staining.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilution

    Lane 1 : Control
    Lane 2 : Starved in HBSS for 2 hours
    Lane 3 : Starved in HBSS for 4 hours
    Lane 4 : Starved in HBSS for 8 hours
    Lane 5 : Starved in HBSS 8 hours + 200 nM Baf A1
    Lane 6 : Starved in HBSS 8 hours + 4 uM Mg132

    Lysates/proteins at 40 µg per lane.

    Secondary
    HRP conjugated goat anti-mouse polyclonal at 1/5000 dilution
    Developed using the ECL technique

    Observed band size : 62 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 seconds

    Image courtesy of Dr Randal Tibbetts by Abreview.

    All lanes are whole cell lysate prepared from HeLa cells. Treatments are listed.

    See Abreview

  • All lanes : Anti-SQSTM1 / p62 antibody (ab56416) at 1/1000 dilution

    Lane 1 : Whole cell lysates prepared from Tzb-naive SKBR3 parental cells.
    Lane 2 : Whole cell lysates prepared from Tzb-refractory TzbR POOL1 cells.
    Lane 3 : Whole cell lysates prepared from Tzb-refractory TzbR POOL2 cells.

    Lysates/proteins at 50 µg per lane.

    Secondary
    Horseradish peroxidase-conjugated secondary
    Developed using the ECL technique

    Image from Vazquez-Martin A et al, PLoS One. 2009 Jul 16;4(7):e6251, Fig 4.

    Cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton® X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonylfluoride, and complete protease inhibitor cocktail for 30 minutes on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 minutes at 14,000×g, 4°C). Protein content was determined against a standardized control using the Pierce Protein Assay Kit. Equal amounts of protein were resuspended in 5× Laemmli sample buffer (10 minutes at 70°C), resolved by electrophoresis on 10% SDS-PAGE, and transferred onto nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 hour at room temperature with TBS-T buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] containing 5% (w/v) nonfat dry milk. The treated filters were washed in TBS-T and then incubated with the primary

  • Developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 62 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 seconds

    This image is courtesy of an anonymous Abreview

    Western blot analysis of Rhesus monkey retinal pigmented epithelium whole cell lysate (20µg/lane) treated with increasing concrentration of an autophagy inhibitor. SQSTM1 / p62 was labelling with ab56416 at 1/1000. An Alkaline Phosphatase-conjugated rabbit anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

    See Abreview

References for Anti-SQSTM1 / p62 antibody (ab56416)

This product has been referenced in:
  • Chen Y  et al. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell. Biochem Biophys Res Commun N/A:N/A (2016). Read more (PubMed: 26993162) »
  • Liang D  et al. Therapeutic efficacy of apelin on transplanted mesenchymal stem cells in hindlimb ischemic mice via regulation of autophagy. Sci Rep 6:21914 (2016). WB ; Mouse . Read more (PubMed: 26902855) »

See all 47 Publications for this product

Product Wall

Application Immunoprecipitation
Sample Mouse Cell lysate - whole cell (Cardiomyocytes)
Total protein in input 100 µg
Immuno-precipitation step Other - Dynabeads
Specification Cardiomyocytes
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Submitted Nov 17 2016

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Application Immunocytochemistry
Sample Mouse Cultured Cells (Cardiomyocytes)
Permeabilization Yes - Triton x-100, 0.01%
Specification Cardiomyocytes
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative Paraformaldehyde
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Submitted Oct 20 2016

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Application Western blot
Sample Human Cell lysate - whole cell (293T)
Gel Running Conditions Non-reduced Non-Denaturing (Native)
Loading amount 40 µg
Specification 293T
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
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Submitted Sep 05 2016

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Application Western blot
Sample Mouse Cell lysate - whole cell (primary macrophage cells)
Gel Running Conditions Reduced Denaturing (4%-16%)
Loading amount 40 µg
Specification primary macrophage cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Jun 28 2016

Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Heart)
Permeabilization Yes - Triton x-100, 0.01%
Specification Heart
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative Paraformaldehyde
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Submitted Apr 21 2016

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Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse Embryonic Fibroblasts)
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris)
Loading amount 20 µg
Treatment minus/plus 200 nM Bafilomycin A1 for 5 hrs
Specification Mouse Embryonic Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
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Submitted Mar 11 2016

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample Chinese Hamster Cell (CHO cell)
Specification CHO cell
Permeabilization Yes - 1% TRITON-X-100
Fixative Paraformaldehyde
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Submitted Mar 18 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample Rat Cell (h9c2)
Specification h9c2
Permeabilization Yes - 1% TRITON-X-100
Fixative Paraformaldehyde
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Submitted Mar 18 2015

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Chinese Hamster Cell lysate - whole cell (CHO cell)
Specification CHO cell
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
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Submitted Nov 03 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Tissue lysate - whole (Brain)
Specification Brain
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Sep 03 2014

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