Anti-FANCD2 antibody [EPR2279(3)]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

Intracellular flow cytometric analysis of permeabilized HeLa cells labeling FANCD2 with ab178705 at 1/10 dilution (red) compared toa rabbit IgG negative control (green).

• WB

Lab

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

Western blot : Anti-FANCD2 antibody [EPR2279(3)] (ab178705) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab178705 was shown to bind specifically to FANCD2. A band was observed at 160 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in FANCD2 knockout cell line. To generate this image, wild-type and FANCD2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (ab178705) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

FANCD2 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HeLa ab255448 cell lysate at 20 µg

Lane 4:

FANCD2 knockout HeLa <a href='/ko/products/cell-lines/human-fancd2-knockout-hela-cell-line-ab261743'>ab261743</a> cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 160 kDa

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• WB

Lab

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

Lanes 1 - 4 : Merged signal (red and green). Green - ab178705 observed at 166 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab178705 was shown to specifically react with FANCD2 when FANCD2 knockout samples were used. Wild-type and FANCD2 knockout samples were subjected to SDS-PAGE. ab178705 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (ab178705) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

FANCD2 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HEK293 cell lysate at 20 µg

Predicted band size: 166 kDa

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• WB

Lab

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

Lanes 1- 2 : Merged signal (red and green). Green - ab178705 observed at 166 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab178705 was shown to react with FANCD2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261743 (knockout cell lysate ab257173) was used. Wild-type HeLa and FANCD2 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. ab178705 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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-FANCD2 antibody [EPR2279(3)] (ab178705) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

FANCD2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human FANCD2 knockout HeLa cell line (<a href='/ko/products/cell-lines/human-fancd2-knockout-hela-cell-line-ab261743'>ab261743</a>)

Predicted band size: 166 kDa

Observed band size: 166 kDa

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• WB

Supplier Data

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (ab178705) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg with FANCD2 (pS1418) peptide

Lane 2:

HeLa cell lysate at 10 µg with Non-phospho peptide

Lane 3:

HeLa cell lysate at 10 µg

Predicted band size: 166 kDa

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• WB

Supplier Data

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (AB178705)

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2279(3)] (ab178705) at 1/1000 dilution

Lane 1:

HeLa cell lysate untreated at 10 µg

Lane 2:

HeLa cell lysate treated with Alkaline Phosphatase at 10 µg

Lane 3:

HeLa cell lysate treated with Lambda Phosphatase at 10 µg

Predicted band size: 166 kDa

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