Recombinant Anti-SQSTM1 / p62 antibody [EPR23101-103] (ab240635)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23101-103] to SQSTM1 / p62
- Suitable for: Flow Cyt (Intra), WB, ICC/IF, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-SQSTM1 / p62 antibody [EPR23101-103]
See all SQSTM1 / p62 primary antibodies -
Description
Rabbit monoclonal [EPR23101-103] to SQSTM1 / p62 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), WB, ICC/IF, IPmore details
Unsuitable for: IHC-P -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: U-2 OS, HeLa, MEF, NIH/3T3, PC-12, C2C12 and J774A.1 lysates. ICC/IF: HeLa, U-2 OS and MEF cells. Flow Cyt (intra): HeLa and MEF cells. IP: HeLa, U-2 OS and MEF cells.
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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
EPR23101-103 -
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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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab240635 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) |
1/500.
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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 |
Use a concentration of 1 µg/ml.
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IP |
1/30.
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Notes |
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Flow Cyt (Intra)
1/500. |
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
1/30. |
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
- Entrez Gene: 18412 Mouse
- Entrez Gene: 113894 Rat
- Omim: 601530 Human
- SwissProt: Q13501 Human
- SwissProt: Q64337 Mouse
- SwissProt: O08623 Rat
- Unigene: 709030 Human
see all -
Alternative names
- A170 antibody
- DMRV antibody
- EBI 3 associated protein of 60 kDa antibody
see all
Images
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ab240635 staining SQSTM1/p62 (autophagosome) in control HeLa cells (left panel) and SQSTM1/p62 in HeLa cells treated with 100nM bafilomycin A1 (ab120497) for 18hrs (right panel). The cells were fixed with 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 ab240635 at 2ug/ml and ab7291 (Tubulin) at 1/1000 dilution 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) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
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ab240635 staining SQSTM1 in wild-type Hap1 cells and SQSTM1 knockout Hap1 cells treated with chloroquine (ab142116, 50μM for 24 hrs). The cells were fixed with 100% methanol (5 min) then 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 ab240635 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution 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) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). 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 [EPR23101-103] (ab240635) 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 kDaab240635 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 ab240635 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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ab240635 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 ab240635 at 1/500 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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Immunoprecipitation of SQSTM1 in U-2 OS cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab240635 pre-coupled to prot.A-Sepharose beads. Samples were washed and processed for western blot with ab56416 at 1/5000. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate. 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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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (Mouse embryo fibroblast) cells labelling SQSTM1 / p62 with ab240635 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in MEF cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.
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SQSTM1 / p62 was immunoprecipitated from 0.35 mg MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate with ab240635 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240635 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: MEF whole cell lysate 10ug
Lane 2: ab240635 IP in MEF whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab240635 in MEF whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 min.
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Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized MEF (Mouse embryonic fibroblast (immortalized)) cells labelling SQSTM1 / p62 with ab240635 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
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All lanes : Anti-SQSTM1 / p62 antibody [EPR23101-103] (ab240635) at 1/1000 dilution
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 5 : C2C12 (mouse myoblasts myoblast), whole cell lysate
Lane 6 : J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate
Lysates/proteins at 20 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?Blocking and dilution buffer: 5% NFDM/TBST.
Exposure times.
Lanes 1-4:3 minutes; Lanes 5-6:92 seconds.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 24086455).
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SQSTM1 / p62 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab240635 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240635 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: HeLa whole cell lysate 10ug
Lane 2: ab240635 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab240635 in HeLa whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.
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Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling SQSTM1 / p62 with ab240635 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling SQSTM1 / p62 with ab240635 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (9)
ab240635 has been referenced in 9 publications.
- Jia M et al. Inhibition of PI3K/AKT/mTOR Signalling Pathway Activates Autophagy and Suppresses Peritoneal Fibrosis in the Process of Peritoneal Dialysis. Front Physiol 13:778479 (2022). PubMed: 35309056
- Zhou W et al. Exercise-induced FNDC5/irisin protects nucleus pulposus cells against senescence and apoptosis by activating autophagy. Exp Mol Med 54:1038-1048 (2022). PubMed: 35882943
- Zhang L et al. Metformin Inhibits HaCaT Cell Proliferation Under Hyperlipidemia Through Reducing Reactive Oxygen Species via FOXO3 Activation. Clin Cosmet Investig Dermatol 15:1403-1413 (2022). PubMed: 35910506
- Huang L et al. Berberine Attenuates IL-1β-Induced Damage of Nucleus Pulposus Cells via Activating the AMPK/mTOR/Ulk1 Pathway. Biomed Res Int 2022:6133629 (2022). PubMed: 35915801
- Shao Z et al. RNA-binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration. Cell Prolif 54:e12975 (2021). PubMed: 33372336
- Xue P et al. Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response. Exp Mol Med 53:667-680 (2021). PubMed: 33864025
- Chen J et al. Inhibition of Autophagy Prevents Panax Notoginseng Saponins (PNS) Protection on Cardiac Myocytes Against Endoplasmic Reticulum (ER) Stress-Induced Mitochondrial Injury, Ca2+ Homeostasis and Associated Apoptosis. Front Pharmacol 12:620812 (2021). PubMed: 33762943
- Tao J et al. Gastrodin induces lysosomal biogenesis and autophagy to prevent the formation of foam cells via AMPK-FoxO1-TFEB signalling axis. J Cell Mol Med 25:5769-5781 (2021). PubMed: 33973365
- Wei L et al. Ginkgetin alleviates high glucose-evoked mesangial cell oxidative stress injury, inflammation, and extracellular matrix (ECM) deposition in an AMPK/mTOR-mediated autophagy axis. Chem Biol Drug Des 98:620-630 (2021). PubMed: 34148304