702670

antibody from Invitrogen Antibodies

Targeting: NBN AT-V1, AT-V2, ATV, NBS, NBS1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

702670

Provider

Invitrogen Antibodies

Product name

NBS1 Recombinant Rabbit Monoclonal Antibody (9H4L65)

Antibody type

Monoclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

9H4L65

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of NBS1 was achieved by transfecting HeLa cells with NBS1 specific siRNA (Silencer® select Product # s9291 and Product # 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 Monoclonal Antibody (Product # 702670, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 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)

Main image

Experimental details

Western blot analysis was performed on Modified Whole cell extracts (1% SDS) (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), LNCaP (Lane 3), MCF-7 (Lane 4), K-562 (Lane 5) and A-431 (Lane 6). The blots were probed with Anti-Nibrin Recombinant Rabbit Monoclonal Antibody (Product # 702670, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 85 kDa band corresponding to Nibrin was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), 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)

Main image

Experimental details

Western blot analysis was performed on Modified Whole cell extracts (1% SDS) (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), LNCaP (Lane 3), MCF-7 (Lane 4), K-562 (Lane 5) and A-431 (Lane 6). The blots were probed with Anti-Nibrin Recombinant Rabbit Monoclonal Antibody (Product # 702670, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 85 kDa band corresponding to Nibrin was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), 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)

Main image

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 Rabbit Monoclonal Antibody (9H4L65)(Product # 702670) using 2 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.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, A549 cells were fixed and permeabilized for detection of endogenous NBS1 using Anti- NBS1 Recombinant Rabbit Monoclonal Antibody (Product # 702670, 5 µg/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 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. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.