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RabMAb

Anti-ATG9A 抗体 [EPR2450(2)] (ab108338)

製品の概要

  • 製品名
    Anti-ATG9A antibody [EPR2450(2)]
    ATG9A 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [EPR2450(2)] to ATG9A
  • 由来種
    Rabbit
  • 特異性
    The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.
  • アプリケーション
    適用あり: Flow Cyt, WB, IP, IHC-P, ICCmore details
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide corresponding to a region within Human ATG9A.

  • ポジティブ・コントロール
    • HepG2, 293T, A375, cell line lysates Mouse brain and rat brain cell lysates Paraffin-embedded human colon tissue
  • 特記事項

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    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.

    This product is a recombinant rabbit monoclonal antibody.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab108338 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
Flow Cyt 1/20.
WB 1/1000. Predicted molecular weight: 94 kDa.
IP 1/10 - 1/100.
IHC-P 1/50. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.

For unpurified use 1/100 - 1/250.

 

ICC 1/50 - 1/100.

ターゲット情報

  • 機能
    Involved in autophagy and cytoplasm to vacuole transport (Cvt) vesicle formation. Plays a key role in the organization of the preautophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle. Cycles between a juxta-nuclear trans-Golgi network compartment and late endosomes. Nutrient starvation induces accumulation on autophagosomes. Starvation-dependent trafficking requires ULK1, ATG13 and SUPT20H.
  • 配列類似性
    Belongs to the ATG9 family.
  • 細胞内局在
    Cytoplasmic vesicle, autophagosome membrane. Golgi apparatus, trans-Golgi network membrane. Late endosome membrane. Endoplasmic reticulum membrane. Under amino acid starvation or rapamycin treatment, redistributes from a juxtanuclear clustered pool to a dispersed peripheral cytosolic pool. The starvation-induced redistribution depends on ULK1, ATG13, as well as SH3GLB1.
  • Information by UniProt
  • 参照データベース
  • 別名
    • APG9 autophagy 9-like 1 antibody
    • APG9 like 1 antibody
    • APG9-like 1 antibody
    • APG9L1 antibody
    • ATG9 antibody
    • ATG9 autophagy related 9 homolog A antibody
    • ATG9 autophagy related 9 homolog A (S. cerevisiae) antibody
    • ATG9A antibody
    • ATG9A_HUMAN antibody
    • Autophagy 9-like 1 protein antibody
    • Autophagy related 9A antibody
    • Autophagy related protein 9A antibody
    • Autophagy-related protein 9A antibody
    • mATG9 antibody
    • MGD3208 antibody
    • OTTHUMP00000206046 antibody
    • OTTHUMP00000206048 antibody
    • OTTHUMP00000206049 antibody
    • OTTHUMP00000206062 antibody
    see all

画像

  • ab108338 (purified) at 1:20 dilution (2μg) immunoprecipitating ATG9A in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate.

    Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
    Lane 2 (+): ab108338 & HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108338 in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate

    For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution. No band in input lane is due to the boiled lysates
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with purified ab108338 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 100% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with Purified ab108338 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human thyroid carcinoma tissue sections labeling ATG9A with Purified ab108338 at 1:50 dilution (4.12 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

  • Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution (purified) + Mouse spinal cord lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 94 kDa



    Blocking and diluting buffer: 5% NFDM/TBST. 

    The lysates are boiled.

  • Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution (purified) + Rat brain lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 94 kDa



    Blocking and diluting buffer: 5% NFDM/TBST.

    The lysates are boiled.

  • Unpurified ab108338 staining ATG9A in the human cell line HepG2 (human hepatocellular carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/40. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • All lanes : Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/10000 dilution (purified)

    Lane 1 : 293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in non-boiled method
    Lane 2 : 293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in boiled method
    Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in non-boiled method
    Lane 4 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in boiled method

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 94 kDa
    Observed band size: 100 kDa (why is the actual band size different from the predicted?)



    We suggest not to boil the sample after lysis.
    Blocking and diluting buffer: 5% NFDM/TBST

    Exposure time:  
    Left image: 5 seconds
    Right image: 2 seconds

     

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: ATG9A knockout HAP1 cell lysate (20 µg)
    Lane 3: HepG2 cell lysate (20 µg)
    Lane 4: A375 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab108338 observed at 100 and 130 kDa. Red - loading control, ab8245, observed at 37 kDa.

    Unpurified ab108338 was shown to specifically react with ATG9A when ATG9A knockout samples were used. Wild-type and ATG9A knockout samples were subjected to SDS-PAGE. ab108338 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

  • All lanes : Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/1000 dilution (unpurified)

    Lane 1 : HepG2 cell lysate
    Lane 2 : 293T cell lysate
    Lane 3 : A375 cell lysate
    Lane 4 : Mouse brain cell lysate
    Lane 5 : Rat brain cell lysate

    Lysates/proteins at 10 µg per lane.

    Predicted band size: 94 kDa

  • Unpurified ab108338, at 1/100, staining ATG9A in paraffin-embedded Human colon tissue by Immunohistochemistry.

  • Unpurified ab108338 at 1/50 dilution, staining ATG9A in HepG2 cells by Immunofluorescence.

参考文献

This product has been referenced in:
  • Jimenez-Orgaz A  et al. Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy. EMBO J 37:235-254 (2018). Human . Read more (PubMed: 29158324) »
  • Zhou C  et al. Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy. Cell Res 27:184-201 (2017). Read more (PubMed: 27934868) »

See all 14 Publications for this product

レビューと Q&A

Application
Western blot
Sample
Cow Cell lysate - whole cell (Lung)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (4-12)
Loading amount
30 µg
Specification
Lung
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
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投稿 Apr 05 2017

Application
Western blot
Sample
Mouse Cell lysate - whole cell (neuroblastoma)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (4-12)
Loading amount
30 µg
Specification
neuroblastoma
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
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投稿 Apr 05 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (kidney)
Permeabilization
Yes - 0.2% triton x100
Specification
kidney
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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投稿 Mar 24 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
African green monkey Cell (kidney)
Permeabilization
Yes - 0.2% triton x100
Specification
kidney
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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投稿 Mar 24 2017

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - 0.3% triton x100
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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投稿 Mar 24 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (4-12)
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
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投稿 Mar 08 2017

Application
Western blot
Sample
African green monkey Cell lysate - whole cell (Kidney)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (4-12)
Loading amount
30 µg
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
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投稿 Mar 06 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (breast cancer cell line)
Gel Running Conditions
Reduced Denaturing (gel 8%)
Loading amount
20 µg
Specification
breast cancer cell line
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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投稿 Nov 24 2015

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

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