使用敲除细胞株进行验证RabMAb

Anti-APG5L/ATG5抗体[EPR1755(2)] (ab108327)

概述

  • 产品名称Anti-APG5L/ATG5抗体[EPR1755(2)]
    参阅全部 APG5L/ATG5 一抗
  • 描述
    兔单克隆抗体[EPR1755(2)] to APG5L/ATG5
  • 经测试应用适用于: ICC/IF, WB, IP, IHC-Pmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide corresponding to residues in Human APG5L/ATG5.

  • 阳性对照
    • Raji, HeLa, HT-1080, Human fetal kidney, C6, Raw264.7, PC-12 and NIH3T3 cell lysates; Human hepatocellular carcinoma and Human ovarian adenocarcinoma tissue
  • 常规说明

    This product is a recombinant rabbit monoclonal antibody.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

     

    Alternative versions available:

    Anti-APG5L/ATG5 antibody (HRP) [EPR1755(2)] (ab199560)

性能

应用

Our Abpromise guarantee covers the use of ab108327 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
ICC/IF 1/100 - 1/250.
WB 1/1000 - 1/10000. Predicted molecular weight: 32 kDa.
IP 1/10 - 1/100.
IHC-P 1/100 - 1/250. Antigen retrieval is recommended.

靶标

  • 功能Involved in autophagic vesicle formation. Conjugation with ATG12, through a ubiquitin-like conjugating system involving ATG7 as an E1-like activating enzyme and ATG10 as an E2-like conjugating enzyme, is essential for its function. The ATG12-ATG5 conjugate acts as an E3-like enzyme which is required for lipidation of ATG8 family proteins and their association to the vesicle membranes. Involved in mitochondrial quality control after oxidative damage, and in subsequent cellular longevity. The ATG12-ATG5 conjugate also negatively regulates the innate antiviral immune response by blocking the type I IFN production pathway through direct association with RARRES3 and MAVS. Also plays a role in translation or delivery of incoming viral RNA to the translation apparatus. Plays a critical role in multiple aspects of lymphocyte development and is essential for both B and T lymphocyte survival and proliferation. Required for optimal processing and presentation of antigens for MHC II. Involved in the maintenance of axon morphology and membrane structures, as well as in normal adipocyte differentiation. Promotes primary ciliogenesis through removal of OFD1 from centriolar satellites and degradation of IFT20 via the autophagic pathway.
    May play an important role in the apoptotic process, possibly within the modified cytoskeleton. Its expression is a relatively late event in the apoptotic process, occurring downstream of caspase activity. Plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.
  • 组织特异性Ubiquitous. The mRNA is present at similar levels in viable and apoptotic cells, whereas the protein is dramatically highly expressed in apoptotic cells.
  • 序列相似性Belongs to the ATG5 family.
  • 翻译后修饰Conjugated to ATG12; which is essential for autophagy, but is not required for association with isolation membrane.
    Acetylated by EP300.
  • 细胞定位Cytoplasm. Preautophagosomal structure membrane. Colocalizes with nonmuscle actin. The conjugate detaches from the membrane immediately before or after autophagosome formation is completed (By similarity). Localizes also to discrete punctae along the ciliary axoneme and to the base of the ciliary axoneme.
  • Information by UniProt
  • 数据库链接
  • 别名
    • APG 5 antibody
    • APG 5L antibody
    • APG5 antibody
    • APG5 autophagy 5 like antibody
    • APG5 like antibody
    • APG5-like antibody
    • APG5L antibody
    • Apoptosis specific protein antibody
    • Apoptosis-specific protein antibody
    • ASP antibody
    • ATG 5 antibody
    • Atg5 antibody
    • ATG5 autophagy related 5 homolog antibody
    • ATG5_HUMAN antibody
    • Autophagy protein 5 antibody
    • hAPG5 antibody
    • Homolog of S Cerevisiae autophagy 5 antibody
    • OTTHUMP00000040507 antibody
    see all

Anti-APG5L/ATG5 antibody [EPR1755(2)] 图像



  • Predicted band size : 32 kDa

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: APG5L/ATG5 knockout HAP1 cell lysate (20 µg)
    Lane 3: Raji cell lysate (20 µg)
    Lane 4: Jeg-3 cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab108327 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.
    ab108327 was shown to specifically react with APG5L/ATG5 when APG5L/ATG5 knockout samples were used. Wild-type and APG5L/ATG5 knockout samples were subjected to SDS-PAGE. ab108327 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) ab216776 secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

  • Unpurified ab108327, at 1/100 dilution, staining APG5L/ATG5 in paraffin-embedded Human ovarian adenocarcinoma tissue by Immunohistochemistry.

  • Immunofluorescence staining of MCF7 cells with purified ab108327 at a working dilution of 1/150, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 100% methanol and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab108327 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

  • All lanes : Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327) at 1/10000 dilution (purified)

    Lane 1 : C6 cell lysate
    Lane 2 : PC-12 cell lysate
    Lane 3 : NIH/3T3 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size : 32 kDa
    Observed band size : 55 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327) at 1/10000 dilution (purified) + Raji cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/10000 dilution

    Predicted band size : 32 kDa
    Observed band size : 55 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327) at 1/2000 dilution (purified) + HT-1080 cell lysate at 20 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size : 32 kDa
    Observed band size : 55 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327) at 1/2000 dilution (purified) + human fetal kidney at 10 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size : 32 kDa
    Observed band size : 32,55 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab180327 at a working dilution of 1/150. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
  • ab180327 (purified) at 1/20 immunoprecipitating CRSP8 in 10 μg PC-12 whole cell lysate (Lanes 1 and 2, observed at 55 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP (HRP) (ab131366) was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST
  • All lanes : Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327) at 1/1000 dilution (unpurified)

    Lane 1 : Raji cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HT-1080 cell lysate
    Lane 4 : Human fetal kidney cell lysate
    Lane 5 : C6 cell lysate
    Lane 6 : Raw264.7 cell lysate
    Lane 7 : PC-12 cell lysate
    Lane 8 : NIH3T3 cell lysate

    Lysates/proteins at 10 µg per lane.


    Predicted band size : 32 kDa

    Secondary antibody - anti-rabbit HRP (ab6721)

  • Unpurified ab108327, at 1/100 dilution, staining APG5L/ATG5 in paraffin-embedded Human hepatocellular carcinoma tissue by Immunohistochemistry.

Anti-APG5L/ATG5 antibody [EPR1755(2)] (ab108327)参考文献

This product has been referenced in:
  • Gómez-Puerto MC  et al. Autophagy proteins ATG5 and ATG7 are essential for the maintenance of human CD34(+) hematopoietic stem-progenitor cells. Stem Cells N/A:N/A (2016). Read more (PubMed: 26930546) »
  • Chen Y  et al. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell. Biochem Biophys Res Commun N/A:N/A (2016). WB ; Human . Read more (PubMed: 26993162) »

See all 12 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Hepato carcinoma HepG2 cells)
Gel Running Conditions Reduced Denaturing (4 - 15%)
Loading amount 20 µg
Specification Hepato carcinoma HepG2 cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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提交于 Nov 17 2016

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (macrophage)
Specification macrophage
Treatment 0.1 ug/ml LPS 24 h
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Verified customer

提交于 Dec 12 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Non-reduced Denaturing (12)
Sample Xenopus tropicalis Tissue lysate - whole (Brain)
Specification Brain
Treatment protease inhibitor cocktail treatment in RIPA lysis buffer
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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提交于 Mar 24 2014

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