Anti-FANCD2 antibody [EPR2302]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FANCD2 antibody [EPR2302] (AB108928)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling FANCD2 with purified ab108928 at 1/50 dilution (3.6 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FANCD2 antibody [EPR2302] (AB108928)

ab108928 (unpurified) staining FANCD2 in wild-type HAP1 cells (top panel) and FANCD2 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 minutes), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab108928 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FANCD2 antibody [EPR2302] (AB108928)

ab108928 (unpurified) staining FANCD2 in human U2OS osteosarcoma cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 10% goat serum for 1 hour at 20°C. Samples were incubated with primary antibody (1/300) for 18 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG monoclonal (1/1000) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FANCD2 antibody [EPR2302] (AB108928)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling FANCD2 (red) with ab108928 (purified) at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr^® 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 primary and secondary antibodies.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FANCD2 antibody [EPR2302] (AB108928)

HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for FANCD2 (green) using ab108928 (unpurified) (1/250 dilution) in ICC/IF.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FANCD2 antibody [EPR2302] (AB108928)

Immunohistochemical staining of paraffin-embedded human tonsil tissue using ab108928 (unpurified) at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FANCD2 antibody [EPR2302] (AB108928)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling FANCD2 with purified ab108928 at 1/500 dilution (0.4 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml) dilution. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IP

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Immunoprecipitation - Anti-FANCD2 antibody [EPR2302] (AB108928)

ab108928 (purified) at 1/20 dilution (1ug) immunoprecipitating FANCD2 in HeLa whole cell lysates.
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates 10ug
Lane 2 (+) : ab108928 & HeLa whole cell lysates
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab108928 in HeLa whole cell lysates
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-FANCD2 antibody [EPR2302] (ab108928)

Predicted band size: 166 kDa

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• WB

Lab

Western blot - Anti-FANCD2 antibody [EPR2302] (AB108928)

Western blot : Anti-FANCD2 antibody [EPR2302] (ab108928) 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, ab108928 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 [EPR2302] (ab108928) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human FANCD2 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-fancd2-knockout-hct116-cell-line-ab286588'>ab286588</a>)

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='/en-us/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 [EPR2302] (AB108928)

Lanes 1- 2 : Merged signal (red and green). Green - ab108928 observed at 166 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab108928 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. ab108928 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 [EPR2302] (ab108928) 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='/en-us/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

Lab

Western blot - Anti-FANCD2 antibody [EPR2302] (AB108928)

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2302] (ab108928) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

Lane 4:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 5:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 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: 166 kDa

Observed band size: 165 kDa

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• WB

Lab

Western blot - Anti-FANCD2 antibody [EPR2302] (AB108928)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : FANCD2 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : HEK293 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab108928 (unpurified) observed at 165 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108928 was shown to specifically react with FANCD2 when FANCD2 knockout samples were used. Wild-type and FANCD2 knockout samples were subjected to SDS-PAGE. ab108928 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-FANCD2 antibody [EPR2302] (ab108928)

Predicted band size: 166 kDa

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