Recombinant 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.

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.

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.

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.

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.

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. 

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.

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.

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