Recombinant Anti-SQSTM1 / p62 antibody [RM1079] (ab314504)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit recombinant multiclonal [RM1079] to SQSTM1 / p62
- Suitable for: WB, IP, IHC-P, Flow Cyt (Intra), ICC/IF, IHC-Fr
- Knockout validated
- Reacts with: Mouse, Rat, Human
Overview
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Product name
Anti-SQSTM1 / p62 antibody [RM1079]
See all SQSTM1 / p62 primary antibodies -
Description
Rabbit recombinant multiclonal [RM1079] to SQSTM1 / p62 -
Host species
Rabbit -
Specificity
Unsuitable for mouse and rat IP.
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Tested applications
Suitable for: WB, IP, IHC-P, Flow Cyt (Intra), ICC/IF, IHC-Frmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
This product was produced with the following immunogens:
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers. -
Positive control
- WB: Wild type HAP1, HeLa, HepG2, MCF7, 293T, K-562, MEF, C6, RAW 264.7, PC-12, NIH/3T3, Human kidney, Mouse spleen and Rat spleen tissue lysates. IHC-P: Human breast cancer, Mouse cerebrum, Rat cerebrum and HCT116 cell pellets. IHC-Fr: Mouse cerebrum and Rat cerebrum tissues. ICC/IF: MEF, PC-12 and HAP1 cells. Flow Cyt (intra): HAP1, MEF and PC-12 cells. IP: HepG2 whole cell lysate.
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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. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol (glycerin, glycerine), 59% PBS, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Recombinant Multiclonal -
Clone number
RM1079 -
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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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab314504 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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WB |
1/1000. Predicted molecular weight: 47 kDa.
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IP |
1/30.
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IHC-P |
1/1000 - 1/20000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/5000.
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ICC/IF |
1/500.
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IHC-Fr |
1/50.
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Notes |
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WB
1/1000. Predicted molecular weight: 47 kDa. |
IP
1/30. |
IHC-P
1/1000 - 1/20000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Flow Cyt (Intra)
1/5000. |
ICC/IF
1/500. |
IHC-Fr
1/50. |
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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All lanes : Anti-SQSTM1 / p62 antibody [RM1079] (ab314504) at 1/1000 dilution
Lane 1 : Wild type HAP1 (human chronic myelogenous leukemia cell) whole cell lysate
Lane 2 : SQSTM1 knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 62 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST
Western blot: Anti-SQSTM1 / p62 antibody (ab314504) staining at 1/1000 dilution, shown in green; Mouse Anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20, 000 dilution, shown in red.
In Western blot, ab314504 was shown to bind specifically to SQSTM1 / p62. Target of interest was observed at 62 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in SQSTM1 knockout cell line (lane 2). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged. -
All lanes : Anti-SQSTM1 / p62 antibody [RM1079] (ab314504) at 1/1000 dilution
Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 4 : MEF (mouse embryo fibroblast) whole cell lysate
Lane 5 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 6 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 8 : NIH/3T3 (mouse embryonic fibroblast) 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 diluting buffer and concentration: 5% NFDM/TBST
Exposure time: Lanes 1-4: 15 seconds; Lanes 5-8: 103 seconds -
All lanes : Anti-SQSTM1 / p62 antibody [RM1079] (ab314504) at 1/1000 dilution
Lane 1 : Human kidney tissue lysate
Lane 2 : Mouse spleen tissue lysate
Lane 3 : Rat spleen tissue 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 diluting buffer and concentration: 5% NFDM/TBST
Exposure time: Lane 1: 103 seconds; Lane 2 and 3: 125 seconds -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 antibody [RM1079] (ab314504)
Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling SQSTM1 with ab314504 at 1/5000 (0.101 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection). Positive staining on human breast cancer.The section was incubated with ab314504 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 antibody [RM1079] (ab314504)
Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling SQSTM1 with ab314504 at 1/1000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection). Positive staining on mouse cerebrum.The section was incubated with ab314504 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 antibody [RM1079] (ab314504)
Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling SQSTM1 with ab314504 at 1/1000 (0.505 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection). Positive staining on rat cerebrum.The section was incubated with ab314504 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 antibody [RM1079] (ab314504)
Immunohistochemical analysis of paraffin-embedded (A) Wild-type HCT116 (human colon epithelial) cell pellet (B) SQSTM1 knockout HCT116 (ab266871) cell pellet tissue labeling SQSTM1 with ab314504 at 1/20000 (0.025 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection). Positive staining on (A) Wild-type HCT116 cell pellet, no staining on (B) SQSTM1 knockout HCT116 (ab266871) cell pellet. The section was incubated with ab314504 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins
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Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh) tissue labeling SQSTM1 with ab314504 at 1/50 (10.1 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining on mouse cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab314504 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).
Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.
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Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (fresh) tissue labeling SQSTM1 with ab314504 at 1/50 (10.1 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green). Confocal image showing positive staining on rat cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab314504 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).
Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast cell) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining in MEF cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining in PC-12 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.
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Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized SQSTM1 KO HAP1(SQSTM1 knockout human chronic myelogenous leukemia cell line) cells labelling SQSTM1 with ab314504 at 1/500 (1.01 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining in HAP1 cell line, and negative staining in SQSTM1 KO HAP1 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.
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Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SQSTM1 KO HAP1 (human SQSTM1 knockout chronic myelogenous leukemia near-haploid cell) / parental HAP1 cells labelling SQSTM1 / p62 with ab314504 at 1/5000 dilution (0.01 ug)/Red (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, ab150081) at 1/5000 dilution was used as the secondary antibody.
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Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MEF (mouse embryo fibroblast) cells labelling SQSTM1 / p62 with ab314504 at 1/5000 dilution (0.01 ug)/Red (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, ab150081) at 1/5000 dilution was used as the secondary antibody.
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Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling SQSTM1 / p62 with ab314504 at 1/5000 dilution (0.01 ug)/Red (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, ab150081) at 1/5000 dilution was used as the secondary antibody.
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SQSTM1 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab314504 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314504 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2: ab314504 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab314504 in HepG2 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.Exposure time: 62 seconds
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
References (0)
ab314504 has not yet been referenced specifically in any publications.