Anti-ATAD2 antibody [EPR12730]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATAD2 antibody [EPR12730] (AB176319)

Immunofluorescent staining of HeLa cells labeling ATAD2 using ab176319 at a 1/100 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATAD2 antibody [EPR12730] (AB176319)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ATAD2 (red) with ab176319 at a 1/60 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

• WB

Lab

Western blot - Anti-ATAD2 antibody [EPR12730] (AB176319)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : ATAD2 knockout HAP1 whole cell lysate (20 μg)

Lanes 1 - 2 : Merged signal (red and green). Green - ab176319 observed at 200 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab176319 was shown to specifically react with ATAD2 in wild type cells as signal was lost in ATAD2 knockout cells. Wild-type and ATAD2 knockout samples were subjected to SDS-PAGE. ab176319 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 500 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.

All lanes:

Western blot - Anti-ATAD2 antibody [EPR12730] (ab176319)

Predicted band size: 159 kDa

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• WB

Lab

Western blot - Anti-ATAD2 antibody [EPR12730] (AB176319)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This protein is prone to degradation. To minimize degradation, cells or tissues were lysed immediately after harvest using freshly prepared lysis buffer, followed by immediate gel electrophoresis and transfer to membranes for Western blot analysis.

All lanes:

Western blot - Anti-ATAD2 antibody [EPR12730] (ab176319) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell (frozen) lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell (fresh) lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell (frozen) lysate at 10 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell (fresh) lysate at 10 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

Observed band size: 180 kDa

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Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-ATAD2 antibody [EPR12730] (AB176319)

All lanes:

Western blot - Anti-ATAD2 antibody [EPR12730] (ab176319) at 1/1000 dilution

Lane 1:

HeLa lysate at 10 µg

Lane 2:

MCF-7 lysate at 10 µg

Lane 3:

T47-D lysate at 10 µg

Lane 4:

Saos-2 lysate at 10 µg

Predicted band size: 159 kDa

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• WB

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Western blot - Anti-ATAD2 antibody [EPR12730] (AB176319)

Lanes 1- 2 : Merged signal (red and green). Green - ab176319 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab176319 was shown to react with ATAD2 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264957 (knockout cell lysate ab257359) lane below 200kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and ATAD2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab176319 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATAD2 antibody [EPR12730] (ab176319) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATAD2 knockout HeLa cell lysate at 20 µg

Predicted band size: 159 kDa

Observed band size: 200 kDa

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• WB

Lab

Western blot - Anti-ATAD2 antibody [EPR12730] (AB176319)

Lanes 1- 2 : Merged signal (red and green). Green - ab176319 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab176319 was shown to react with ATAD2 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264957 (CRISPR/Cas9 edited cell lysate ab257359) lane below 200kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and ATAD2 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab176319 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATAD2 antibody [EPR12730] (ab176319) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATAD2 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATAD2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-atad2-knockout-hela-cell-line-ab264957'>ab264957</a>)

Predicted band size: 159 kDa

Observed band size: 200 kDa

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