All lanes : Anti-FANCD2 antibody [EPR2302] (ab108928) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FANCD2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 166 kDa
Observed band size: 166 kDa

This data was developed using the same antibody clone in a different buffer formulation (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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

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 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 primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108928).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108928).

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

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108928).