- 703744 - Invitrogen Antibodies PRMT3 antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of PRMT3 was achieved by transfecting A549 cells with PRMT3 specific validated siRNA (Silencer® select Product # s19870). Western blot analysis (Fig a) was performed using whole cell extracts from PRMT3 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) at a 1:5000 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 PRMT3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) and a 60 kDa band corresponding to PRMT3 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), MDA-MB-231 (Lane 3), MCF7 (Lane 4), T-47D (Lane 5), U-2 OS (Lane 6), LNCaP (Lane 7) and PC-12 (Lane 8) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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 the primary antibody (1:5000 dilution) and detected by chemiluminescence 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)
Main image
Experimental details: For immunocytochemistry analysis, A549 cells were fixed and permeabilized for detection of endogenous PRMT3 using Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) 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 PRMT3 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 cytoplasmic localization of PRMT3. 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)
Main image
Experimental details: Knockdown of PRMT3 was achieved by transfecting A549 cells with specific validated siRNA (Silencer® select Product # s19870). Immunofluorescence analysis was performed on A549 cells (untransfected, panel a-d), transfected with PRMT3 specific siRNA (panel i-l) or non-specific scrambled siRNA (panels e-h). Cells were fixed, permeabilized, and labeled with Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) at a 1:100 dilution, followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a 1:2000 dilution. Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415) at a 1:300 dilution was used for cytoskeletal F-actin (red) staining. Significant reduction of the signal was observed upon siRNA mediated knockdown (panel i-l) confirming the specificity of the antibody to PRMT3 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: PRMT3 was immunoprecipitated using 5 µg of the Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) from whole cell extracts (800µg) of A549 (Lane 3) using the Protein A/G Dynabeads® (Product # 10001D and Product # 10003D). Normal Rabbit IgG was used as a Isotype control (Lane 2). Western blot analysis was performed using Anti-PRMT3 Recombinant Rabbit Monoclonal Antibody (Product # 703744) at a 1:5000 dilution. The blot was detected by chemiluminescence using Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, light chain specific antibody (Product # 211-032-171) at a 1:10000 dilution. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP03011BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

703744