- 711701 - Invitrogen Antibodies NBN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Nuclear extracts (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), LNCap (Lane 3), MCF-7 (Lane 4), K-562 (Lane 5), A-431 (Lane 6) and PC-12 (Lane 7). The blots were probed with Anti-NBS1 Recombinant Rabbit Polyclonal Antibody (Product # 711701, 1 µg/mL). A 85 kDa band corresponding to NBS1 was observed across the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NBS1 was achieved by transfecting HeLa cells with NBS1 specific siRNA (Silencer® select Cat # s9291 and s9292). Western blot analysis (Fig a) was performed using Modified Whole cell extract (1% SDS) from the NBS knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-NBS1 Recombinant Rabbit Polyclonal Antibody (Product # 711701, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to NBS1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of NBN was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042) (Assay ID CRISPR791902_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of NBN was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2), HeLa NBN KO (Lane 3) whole cell extracts. The blot was probed with Anti-NBS1 Recombinant Polyclonal Antibody (Product # 711701) using 1 µg/mL dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to NBN.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, A549 cells were fixed and permeabilized for detection of endogenous NBS1 using Anti- NBS1 Recombinant Rabbit Polyclonal Antibody (Product # 711701, 2µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of NBS1 (Nibrin)/NBN protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating Nuclear localization of NBS1 (Nibrin)/NBN. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

711701