PA1-16548

antibody from Invitrogen Antibodies

Targeting: FANCD2 FA-D2, FACD, FAD, FANCD

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Chromatin Immunoprecipitation

Antibody data

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Validation data

Product number: PA1-16548 - Provider product page
Provider: Invitrogen Antibodies
Product name: FANCD2 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Other
Description: In Western blot, this antibody should recognize a band at ~166 kDa (post-translationally modified form). Additional bands may be seen at lower molecular weights. In immunofluorescence, this has been tested in human MMC and IR-treated MEF cells.Suggested positive control: Hela whole cell extract.
Reactivity: Human, Mouse, Rat, Canine, Chicken/Avian, Zebrafish
Host: Rabbit
Isotype: IgG
Vial size: 50 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

FANCD2 protein structure - PA1-16548 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of FANCD2 in HeLa WCE using Product # PA1-16548 (lot C).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on modified whole cell extracts (1% SDS) (30 µg lysate) of Jurkat (Lane 1), U-2 OS (Lane 2), HeLa (Lane 3), HeLa treated with Hydroxyurea (2mmol for 24 hrs) (Lane 4). The blot was probed with FANCD2 Polyclonal Antibody (Product # PA1-16548, 1:15000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # PA1-16548, A27036, 0.25 µg/ml, 1:4000 dilution). A band at ~166 kDa corresponding to FANCD2 was observed across cell lines tested and was enhanced upon treatment with hydroxyurea in HeLa cell line.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of FANCD2 in HeLa WCE. Sample was incubated in FANCD2 polyclonal antibody (Product # PA1-16548).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of FANCD2 in 0.1 mg/mL HeLa lysate. Samples were incubated in FANCD2 polyclonal antibody (Product # PA1-16548). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of FANCD2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR718017_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of FANCD2 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa FANCD2 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-FANCD2 Polyclonal Antibody (Product # PA1-16548, 1:10000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to FANCD2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of FANCD2 in SiHa cells after cell exposure to IR. Samples were incubated in FANCD2 polyclonal antibody (Product # PA1-16548). Analysis using the Biotin conjugate of FANCD2 Antibody. FANCD2 colocalizes in vivo with another protein in SiHa cells after cell exposure to IR. Proliferating SiHa cells were exposed to 10 Gy of IR and double color immunofluorescence staining was performed after 8 hours. Images were captured in a Kodak digital image system on a Leica fluorescence microscope.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of FANCD2 was performed using 70% confluent log phase HeLa cells treated with 2mM of Hydroxyurea for 24 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with FANCD2 Rabbit Polyclonal Antibody (Product # PA1-16548) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows untreated cells with reduced signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.