Recombinant Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18351] to SQSTM1 / p62
- Suitable for: Flow Cyt (Intra), IHC-P, WB, ICC/IF, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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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 (Intra), IHC-P, WB, ICC/IF, IPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Human liver and kidney lysates; U-2 OS, HCT116, 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), U-2 OS cells. Flow Cyt (intra): PC-3 cells. IP: HeLa and HepG2 whole cell lysates.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18351 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
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 |
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Flow Cyt (Intra) |
1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IHC-P |
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB |
1/1000. Detects a band of approximately 62 kDa (predicted molecular weight: 47 kDa).
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ICC/IF | (1) |
Use a concentration of 1 µg/ml.
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IP | (2) |
1/40.
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Notes |
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Flow Cyt (Intra)
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
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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
modificationsPhosphorylated. 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
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Database links
- Entrez Gene: 8878 Human
- Omim: 601530 Human
- SwissProt: Q13501 Human
- Unigene: 709030 Human
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Alternative names
- A170 antibody
- DMRV antibody
- EBI 3 associated protein of 60 kDa antibody
see all
Images
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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.
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ab207305 was shown to react with SQSTM1 in wild-type U-2 OS cells in Immunocytochemistry with loss of signal observed in a SQSTM1 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1:1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1/5000. The cells were then incubated with ab207305 at 1/200 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
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All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 CRISPR/Cas9 edited HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.
ab207305 Anti-SQSTM1 / p62 antibody [EPR18351] was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in CRISPR/Cas9 edited cell line ab266871 (CRISPR/Cas9 edited cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 CRISPR/Cas9 edited samples were subjected to SDS-PAGE. ab207305 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.
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ab207305 staining SQSTM1 in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (ab142116, 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).
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All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305) at 1/1000 dilution
Lane 1 : Wild-type U-2 OS cell lysate
Lane 2 : SQSTM1 knockout U-2 OS cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDaab207305 was shown to react with SQSTM1 in wild-type U-2 OS cells in Western blot with loss of signal observed in a SQSTM1 knockout cell line. Wild-type U-2 OS and SQSTM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab207305 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2ug/mL before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
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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. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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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).
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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 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).
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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 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Intracellular 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.
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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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (39)
ab207305 has been referenced in 39 publications.
- Takata T et al. Intracellular Toxic Advanced Glycation End-Products in 1.4E7 Cell Line Induce Death with Reduction of Microtubule-Associated Protein 1 Light Chain 3 and p62. Nutrients 14:N/A (2022). PubMed: 35057513
- Sun Z et al. Sec23a inhibits the self-renewal of melanoma cancer stem cells via inactivation of ER-phagy. Cell Commun Signal 20:22 (2022). PubMed: 35236368
- Zhu M et al. AMPK Activator O304 Protects Against Kidney Aging Through Promoting Energy Metabolism and Autophagy. Front Pharmacol 13:836496 (2022). PubMed: 35308246
- Gan Y et al. Estradiol inhibits autophagy of Mycobacterium tuberculosis‑infected 16HBE cells and controls the proliferation of intracellular Mycobacterium tuberculosis. Mol Med Rep 25:N/A (2022). PubMed: 35425995
- Hickman RA et al. The distribution and density of Huntingtin inclusions across the Huntington disease neocortex: regional correlations with Huntingtin repeat expansion independent of pathologic grade. Acta Neuropathol Commun 10:55 (2022). PubMed: 35440014