Validated using a knockout cell line
Recombinant
RabMAb

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)

Overview

  • Product name
    Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
    See all SQSTM1 / p62 primary antibodies
  • Description
    Rabbit monoclonal [EPR4844] to SQSTM1 / p62 - Autophagosome Marker
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, Flow Cyt, ICC/IFmore 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.
    Database link: Q13501

  • Positive control
    • WB: MCF-7; HeLa; SKBR-3; 293T. 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

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

    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 ab109012 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
WB 1/10000 - 1/50000. Detects a band of approximately 62 kDa.
Flow Cyt 1/10 - 1/100.

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

ICC/IF Use a concentration of 1 µg/ml.

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 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 - 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.

  • 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).

  • All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)

    Lane 1 : MCF-7
    Lane 2 : HeLa
    Lane 3 : SKBR-3
    Lane 4 : 293T

    Lysates/proteins at 10 µg per lane.

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

  • Overlay histogram showing HeLa cells stained with 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.

  • 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).

References

This product has been referenced in:
  • Bartolome F  et al. Amyloid ß-induced impairments on mitochondrial dynamics, hippocampal neurogenesis, and memory are restored by phosphodiesterase 7 inhibition. Alzheimers Res Ther 10:24 (2018). WB . Read more (PubMed: 29458418) »
  • Flores-Costa R  et al. The soluble guanylate cyclase stimulator IW-1973 prevents inflammation and fibrosis in experimental non-alcoholic steatohepatitis. Br J Pharmacol 175:953-967 (2018). Read more (PubMed: 29281143) »
See all 45 Publications for this product

Customer reviews and Q&As

1-10 of 15 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (MEF cells)
Total protein in input
500 µg
Immuno-precipitation step
Protein A/G
Specification
MEF cells

Abcam user community

Verified customer

Submitted Nov 30 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
50 µg
Specification
MEF cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 26 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (293T cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
50 µg
Specification
293T cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 29 2018

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (293T cells)
Total protein in input
500 µg
Immuno-precipitation step
Protein A/G
Specification
293T cells

Abcam user community

Verified customer

Submitted Oct 29 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing
Loading amount
40 µg
Specification
HEK293
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 18 2018

Application
Immunocytochemistry
Sample
Human Cell (Hela cells (Epithelial))
Permeabilization
Yes - 0.2% Triron X100
Specification
Hela cells (Epithelial)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Methanol

Abcam user community

Verified customer

Submitted Aug 14 2017

Application
Western blot
Sample
Cow Cell lysate - whole cell (Kidney)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 20 2017

Application
Western blot
Sample
African green monkey Cell lysate - whole cell (Kidney)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 20 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 20 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Brain)
Gel Running Conditions
Reduced Denaturing
Loading amount
15 µg
Specification
Brain
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 25 2017

1-10 of 15 Abreviews or Q&A

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