- MA5-14953 - Invitrogen Antibodies XRCC5 antibody

Eberle J, Wiehe RS, Gole B, Mattis LJ, Palmer A, Ständker L, Forssmann WG, Münch J, Gebhardt JCM, Wiesmüller L
Frontiers in oncology 2021;11:689063

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Ku80 was achieved by transfecting HeLa with Ku80 specific siRNAs (Silencer® select Product # s14952, s14953). Western blot analysis (Fig. a) was performed using whole cell extracts from the Ku80 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Ku80 Monoclonal Antibody (Product # MA5-14953, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Ku80.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Ku80 Monoclonal Antibody (S.669.4), (Product # MA5-14953) and a 80 kDa band corresponding to Ku80 was observed in the cell lines tested. Modified whole cell extracts (1%SDS) (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), K-562 (Lane 3), Jurkat (Lane 4), COS-7 (Lane 5), A549 (Lane 6) and HEK-293 (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Ku80 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Ku80 Monoclonal Antibody (Product # MA5-14953) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant 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 SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing staining in nucleus. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous Ku80 protein using Anti-Ku80 Antibody: ChIP was performed using Anti-Ku80 Rabbit Monoclonal Antibody (Product # MA5-14953, 5 µg) on sheared chromatin from Camptothecin treated HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to GAPDH transcriptional start site, GAPDH gene body (+2Kb), CDKN1A intron 1 and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-14953