Anti-ATG9A antibody [EPR2450(2)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling ATG9A with ab108338 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab108338 anti-ATG9A antibody [EPR2450(2)] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with purified ab108338 at 1/20 dilution (10μg/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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Unpurified ab108338 at 1/50 dilution, staining ATG9A in HepG2 (Human hepatocellular carcinoma epithelial cell) cells by Immunofluorescence.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Unpurified ab108338 staining ATG9Ain the human cell line HepG2 (human hepatocellular carcinoma) by intracellular 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)

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Accumulation of ATG9A at the TGN of AP-4 μ4 mutant patient fibroblasts.

Skin fibroblasts were from one control individual and two patients homozygous for mutations in the AP4M1 gene encoding AP-4 μ4.

Co-immunostaining for endogenous ATG9A (green) and AP-4 ε (red) (B) or GM130 of the fibroblasts.

ATG9A is detected using ab108338.

(From Figure 4B of De Pace et al)

De Pace, R. et al PLoS Genet. 2018 Apr 26;14(4):e1007363. doi: 10.1371/journal.pgen.1007363. eCollection 2018 Apr Reproduced under the Creative Commons license https://creativecommons.org/publicdomain/zero/1.0/

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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.

• IP

Lab

Immunoprecipitation - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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 Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution. No band in input lane is due to the boiled lysates
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ATG9A antibody [EPR2450(2)] (ab108338)

Predicted band size: 94 kDa

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• WB

Lab

Western blot - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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

All lanes:

Western blot - Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/10000 dilution

Lane 1:

293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in non-boiled method at 15 µg

Lane 2:

293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in boiled method at 15 µg

Lane 3:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in non-boiled method at 15 µg

Lane 4:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in boiled method at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 94 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

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:

Western blot - Anti-ATG9A antibody [EPR2450(2)] (ab108338)

Predicted band size: 94 kDa

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• WB

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Western blot - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

All lanes:

Western blot - Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

A375 cell lysate at 10 µg

Lane 4:

Mouse brain cell lysate at 10 µg

Lane 5:

Rat brain cell lysate at 10 µg

Predicted band size: 94 kDa

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• WB

Lab

Western blot - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Blocking and diluting buffer : 5% NFDM/TBST.

The lysates are boiled.

All lanes:

Western blot - Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution

All lanes:

Mouse spinal cord lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 103 kDa,107 kDa,110 kDa,134 kDa,137 kDa,14 kDa,169 kDa,190 kDa,20 kDa,21 kDa,23 kDa,24 kDa,25 kDa,28 kDa,30 kDa,33 kDa,42 kDa,43 kDa,47 kDa,51 kDa,53 kDa,55 kDa,74 kDa,83 kDa,90 kDa,94 kDa,96 kDa,99 kDa

Observed band size: 103 kDa,120 kDa,14 kDa,150 kDa,169 kDa,175 kDa,18 kDa,193 kDa,21 kDa,28 kDa,32 kDa,32-42 kDa,35 kDa,42 kDa,44 kDa,45 kDa,47 kDa,51 kDa,53 kDa,55 kDa,90 kDa

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• WB

Lab

Western blot - Anti-ATG9A antibody [EPR2450(2)] (AB108338)

Blocking and diluting buffer : 5% NFDM/TBST.

The lysates are boiled.

All lanes:

Western blot - Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution

All lanes:

Rat brain lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 42 kDa,47 kDa,53 kDa,76 kDa,82 kDa,94 kDa

Observed band size: 50-70 kDa,53 kDa,72 kDa

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