使用敲除细胞株进行验证RabMAb

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

概述

性能

应用

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.

应用 Ab评论 说明
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 1/100 - 1/500.

靶标

  • 功能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.
  • 组织特异性Ubiquitously expressed.
  • 疾病相关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.
  • 序列相似性Contains 1 OPR domain.
    Contains 1 UBA domain.
    Contains 1 ZZ-type zinc finger.
  • 结构域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.
  • 翻译后修饰Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN.
  • 细胞定位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
  • 数据库链接
  • 别名
    • 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

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker 图像

  • 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 47 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109012 was shown to specifically react with SQSTM1 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 10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Autophagosome Marker with ab109012 at 1/500 dilution (4.6μg/ml). Cells were fixed in 100% Methanol. ab150077 an AlexaFluor® 488 Goat anti-Rabbit secondary antibody was used at 1/1000 dilution (2 μg/ml). DAPI was used to counter stain the nucleus. Confocal image showing cytoplasmic staining on HeLa cell line.

  • 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 of SQSTM1/p62 by immunofluorescence

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

This product has been referenced in:
  • Gentry EG  et al. Rho Kinase Inhibition as a Therapeutic for Progressive Supranuclear Palsy and Corticobasal Degeneration. J Neurosci 36:1316-23 (2016). Read more (PubMed: 26818518) »
  • Fiuza-Luces C  et al. Muscle Signaling in Exercise Intolerance: Insights from the McArdle Mouse Model. Med Sci Sports Exerc N/A:N/A (2016). WB . Read more (PubMed: 27031745) »

See all 5 Publications for this product

Product Wall

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
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提交于 May 25 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Brain)
Permeabilization Yes - 0.3% triton x 100
Specification Brain
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative Paraformaldehyde
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提交于 May 19 2017

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Non-Denaturing (Native) (4-12%)
Sample Human Cell lysate - whole cell (brain gliobastoma from patients)
Specification brain gliobastoma from patients
Blocking step No blocking step used for 12 hour(s) and 0 minute(s) · Temperature: 24°C
Username

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Verified customer

提交于 Jan 13 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated
Sample Rat Tissue sections (ovary)
Specification ovary
Permeabilization No
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Jan 07 2014



We have not tested this antibody specifically for cross-reactivity with ZIP1 or ZIP2, but I ran a BLAST of the ab109012 immunogen sequence against these two ZIP proteins, and there was no significant similarity.

Thank you for contacting us.

The link to the database entry for the human protein is as follows:
http://www.uniprot.org/uniprot/Q13501



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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"