- 703926 - Invitrogen Antibodies SMC3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SMC3 Recombinant Rabbit Monoclonal Antibody (Product # 703926) and a ~140 kDa band corresponding to SMC3 was observed across the cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of MCF7 (Lane 1), HCT 116 (Lane 2), Jurkat (Lane 3), HEK-293 (Lane 4), NIH/3T3 (Lane 5) and C2C12 (Lane 6) were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX). Resolved proteins were then transferred onto a nitrocellulose membrane by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a 1:4000 dilution. 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: Knockdown of SMC3 was achieved by transfecting HeLa cells with SMC3 specific siRNA (Silencer® select Product # s17426 and s17427). Western blot analysis (Fig a) was performed using modified whole cell extracts (1% SDS) from knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-SMC3 Recombinant Rabbit Monoclonal Antibody (Product # 703926) at a 1:1000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) (0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that antibody is specific to SMC3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunocytochemistry analysis, HeLa cells were fixed and permeabilized for detection of endogenous SMC3 using Anti-SMC3 Recombinant Rabbit Monoclonal Antibody (Product # 703926) at a 1:100 dilution and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a 1:2000 dilution. Panel a) shows representative cells that were stained for detection and localization of SMC3 protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415) at a 1:300 dilution. Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of SMC3. 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 SMC3 protein using Anti-SMC3 Antibody: ChIP was performed using 5 µg of Anti-SMC3 Recombinant Rabbit Monoclonal Antibody (Product # 703926) on sheared chromatin from 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 selected chromosomal positions: Chr 14 (75955167-75955445), Chr 17 (7944729-794507), Chr 12 (53746435-53746713), Chr 9 (11244 - 11320) and and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

703926