Recombinant 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.

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)

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.

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). 

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.

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

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.

Blocking and diluting buffer: 5% NFDM/TBST. 

The lysates are boiled.

Blocking and diluting buffer: 5% NFDM/TBST.

The lysates are boiled.

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) 

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.

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