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SQSTM1 (p62) Antibody (C-term)

Purified Rabbit Polyclonal Antibody (Pab)

Immunofluorescence staining of Autophagy SQSTM1 (p62) Antibody (C-term) (Cat# AP2183b) on Methanol-fixed and PFA fixed HeLa cells. Data courtesy of Dr. Eeva-Liisa Eskelinen, University of Helsinki,Finland.
Fluorescent image of U251 cells stained with SQSTM1 (p62) (C-term) antibody. U251 cells were treated with Chloroquine (50 μM,16h), then fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with AP2183b SQSTM1 (p62) (C-term) primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor® 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 μg/ml, 5 min). SQSTM1 (p62) immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells, supported by Human Protein Atlas Data (http://www.proteinatlas.org/ENSG00000161011).
Formalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. BC = breast carcinoma; HC = hepatocarcinoma.
All lanes : Anti-SQSTM1 (p62) Antibody (C-term) at 1:2000 dilution Lane 1: A549 whole cell lysate Lane 2: C2C12 whole cell lysate Lysates/proteins at 20 µg per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 48 kDa Blocking/Dilution buffer: 5% NFDM/TBST.
Western blot analysis of SQSTM1 Antibody (C331) Pab (Cat.#AP2183b) pre-incubated without(lane 1) and with(lane 2) blocking peptide in MCF-7 cell line lysate. SQSTM1 Antibody (C331) (arrow) was detected using the purified Pab.
Western blot analysis of SQSTM1 (arrow) using rabbit polyclonal Autophagy SQSTM1 (p62) Antibody (C-term ) (Cat.#AP2183b). 293 cell lysates (2 ug/lane) either nontransfected (Lane 1) or transiently transfected with the SQSTM1 gene (Lane 2) (Origene Technologies).

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Product Information
Application Applications Legend: E=ELISA WB=Western Blotting IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin) IP=Immunoprecipitation IF=Immunofluorescence IC=Immunochemistry ICC=Immunocytochemistry FC=Flow Cytometry DB=Dot Blot	WB, IHC-P, IF, E
Primary Accession	Q13501
Other Accession	O08623, Q64337
Reactivity	Human, Mouse, Rat
Predicted	Mouse, Rat
Host	Rabbit
Clonality	Polyclonal
Isotype	Rabbit IgG
Calculated MW	47687 Da
Antigen Region	317-346 aa

Additional Information
Gene ID	8878
Other Names	Sequestosome-1, EBI3-associated protein of 60 kDa, EBIAP, p60, Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa, Ubiquitin-binding protein p62, SQSTM1, ORCA, OSIL.
Target/Specificity	This SQSTM1 (p62) antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 317-346 amino acids of human SQSTM1 (p62).
Dilution	WB~~1:1000 IHC-P~~1:100~500 IF~~1:200 E~~Use at an assay dependent concentration.
Format	Purified polyclonal antibody supplied in PBS with 0.09% (W/V) sodium azide. This antibody is prepared by Saturated Ammonium Sulfate (SAS) precipitation followed by dialysis against PBS.
Storage	Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
Precautions	SQSTM1 (p62) Antibody (C-term) is for research use only and not for use in diagnostic or therapeutic procedures.

Protein Information
Name	SQSTM1 {ECO:0000303\|PubMed:16286508, ECO:0000312\|HGNC:HGNC:11280}
Function	Molecular adapter required for selective macroautophagy (aggrephagy) by acting as a bridge between polyubiquitinated proteins and autophagosomes (PubMed:15340068, PubMed:15953362, PubMed:16286508, PubMed:17580304, PubMed:20168092, PubMed:22017874, PubMed:22622177, PubMed:24128730, PubMed:28404643, PubMed:29343546, PubMed:29507397, PubMed:31857589, PubMed:33509017, PubMed:34471133, PubMed:34893540, PubMed:35831301, PubMed:37306101, PubMed:37802024). Promotes the recruitment of ubiquitinated cargo proteins to autophagosomes via multiple domains that bridge proteins and organelles in different steps (PubMed:16286508, PubMed:20168092, PubMed:22622177, PubMed:24128730, PubMed:28404643, PubMed:29343546, PubMed:29507397, PubMed:34893540, PubMed:37802024). SQSTM1 first mediates the assembly and removal of ubiquitinated proteins by undergoing liquid-liquid phase separation upon binding to ubiquitinated proteins via its UBA domain, leading to the formation of insoluble cytoplasmic inclusions, known as p62 bodies (PubMed:15911346, PubMed:20168092, PubMed:22017874, PubMed:24128730, PubMed:29343546, PubMed:29507397, PubMed:31857589, PubMed:37802024). SQSTM1 then interacts with ATG8 family proteins on autophagosomes via its LIR motif, leading to p62 body recruitment to autophagosomes, followed by autophagic clearance of ubiquitinated proteins (PubMed:16286508, PubMed:17580304, PubMed:20168092, PubMed:22622177, PubMed:24128730, PubMed:28404643, PubMed:37802024). SQSTM1 is itself degraded along with its ubiquitinated cargos (PubMed:16286508, PubMed:17580304, PubMed:37802024). Also required to recruit ubiquitinated proteins to PML bodies in the nucleus (PubMed:20168092). Also involved in autophagy of peroxisomes (pexophagy) in response to reactive oxygen species (ROS) by acting as a bridge between ubiquitinated PEX5 receptor and autophagosomes (PubMed:26344566). Acts as an activator of the NFE2L2/NRF2 pathway via interaction with KEAP1: interaction inactivates the BCR(KEAP1) complex by sequestering the complex in inclusion bodies, promoting nuclear accumulation of NFE2L2/NRF2 and subsequent expression of cytoprotective genes (PubMed:20452972, PubMed:28380357, PubMed:33393215, PubMed:37306101). Promotes relocalization of 'Lys-63'-linked ubiquitinated STING1 to autophagosomes (PubMed:29496741). Involved in endosome organization by retaining vesicles in the perinuclear cloud: following ubiquitination by RNF26, attracts specific vesicle-associated adapters, forming a molecular bridge that restrains cognate vesicles in the perinuclear region and organizes the endosomal pathway for efficient cargo transport (PubMed:27368102, PubMed:33472082). Sequesters tensin TNS2 into cytoplasmic puncta, promoting TNS2 ubiquitination and proteasomal degradation (PubMed:25101860). May regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1 (PubMed:10356400, PubMed:10747026, PubMed:11244088, PubMed:12471037, PubMed:16079148, PubMed:19931284). May play a role in titin/TTN downstream signaling in muscle cells (PubMed:15802564). Adapter that mediates the interaction between TRAF6 and CYLD (By similarity).
Cellular Location	Cytoplasmic vesicle, autophagosome. Preautophagosomal structure. Cytoplasm, cytosol. Nucleus, PML body. Late endosome. Lysosome. Nucleus Endoplasmic reticulum. Cytoplasm, myofibril, sarcomere {ECO:0000250\|UniProtKB:O08623}. Note=In cardiac muscle, localizes to the sarcomeric band (By similarity). Localizes to cytoplasmic membraneless inclusion bodies, known as p62 bodies, containing polyubiquitinated protein aggregates (PubMed:11786419, PubMed:20357094, PubMed:22017874, PubMed:29343546, PubMed:29507397, PubMed:31857589, PubMed:37306101, PubMed:37802024). In neurodegenerative diseases, detected in Lewy bodies in Parkinson disease, neurofibrillary tangles in Alzheimer disease, and HTT aggregates in Huntington disease (PubMed:15158159). In protein aggregate diseases of the liver, found in large amounts in Mallory bodies of alcoholic and nonalcoholic steatohepatitis, hyaline bodies in hepatocellular carcinoma, and in SERPINA1 aggregates (PubMed:11981755) Enriched in Rosenthal fibers of pilocytic astrocytoma (PubMed:11786419). In the cytoplasm, observed in both membrane-free ubiquitin-containing protein aggregates (sequestosomes) and membrane- surrounded autophagosomes (PubMed:15953362, PubMed:17580304) Colocalizes with TRIM13 in the perinuclear endoplasmic reticulum (PubMed:22178386). Co-localizes with TRIM5 in cytoplasmic bodies (PubMed:20357094). When nuclear export is blocked by treatment with leptomycin B, accumulates in PML bodies (PubMed:20168092) {ECO:0000250\|UniProtKB:O08623, ECO:0000269\|PubMed:11786419, ECO:0000269\|PubMed:11981755, ECO:0000269\|PubMed:15158159, ECO:0000269\|PubMed:15953362, ECO:0000269\|PubMed:17580304, ECO:0000269\|PubMed:20168092, ECO:0000269\|PubMed:20357094, ECO:0000269\|PubMed:22017874, ECO:0000269\|PubMed:22178386, ECO:0000269\|PubMed:29343546, ECO:0000269\|PubMed:29507397, ECO:0000269\|PubMed:31857589, ECO:0000269\|PubMed:37306101, ECO:0000269\|PubMed:37802024}
Tissue Location	Ubiquitously expressed.

Research Areas

For Research Use Only. Not For Use In Diagnostic Procedures.

Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol

BACKGROUND

SQSTM1/p62 is an adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. This protein may play a role in titin/TTN downstream signaling in muscle cells, and may also regulate signaling cascades through ubiquitination. This protein is involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels. SQSTM1/p62 also appears to play a role in macroautophagic removal of intracellular protein aggregates. Cellular depletion studies of SQSTM1/p62 have indicated a role for association with LC3 and aggregate proteins in order to facilitate normal formation of the autophagosome.

REFERENCES

References for protein:
1.Seibenhener, M.L., et al., Mol. Cell. Biol. 24(18):8055-8068 (2004).
2.Eekhoff, E.W., et al., Arthritis Rheum. 50(5):1650-1654 (2004).
3.Brajenovic, M., et al., J. Biol. Chem. 279(13):12804-12811 (2004).
4.Kuusisto, E., et al., J. Neuropathol. Exp. Neurol. 62(12):1241-1253 (2003).
5. Johnson-Pais, T.L., et al., J. Bone Miner. Res. 18(10):1748-1753 (2003).
References for U251 cell line:
1. Westermark B.; Pontén J.; Hugosson R. (1973).” Determinants for the establishment of permanent tissue culture lines from human gliomas”. Acta Pathol Microbiol Scand A. 81:791-805. [PMID: 4359449].
2. Pontén, J.,Westermark B. (1978).” Properties of Human Malignant Glioma Cells in Vitro”. Medical Biology 56: 184-193.[PMID: 359950].
3. Geng Y.;Kohli L.; Klocke B.J.; Roth K.A.(2010). “Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent”. Neuro Oncol. 12(5): 473–481.[ PMID: 20406898].

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Cat# AP2183B

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SQSTM1 (p62) Antibody (C-term)

Purified Rabbit Polyclonal Antibody (Pab)

BACKGROUND

REFERENCES

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