703794

antibody from Invitrogen Antibodies

Targeting: GOLGA2 GM130, golgin-95

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

703794

Provider

Invitrogen Antibodies

Product name

GM130 Recombinant Rabbit Monoclonal Antibody (10H5L5)

Antibody type

Monoclonal

Antigen

Other

Description

This antibody is predicted to react with Mouse, Rat, Bovine.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

10H5L5

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

Western blot was performed using Anti-GM130 Recombinant Rabbit Monoclonal Antibody (Product # 703794) and a 140 kDa band corresponding to was GM130 served across the cell lines tested. Whole cell extract (30 µg lysate) of HeLa (Lane 1), MDA-MB-231(Lane 2) and U-2 OS (Lane 3) were electrophoresed using Novex® NuPAGE™ 4-12% Bis-Tris Protein 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 Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # A27036, 1:5000 dilution) using the iBright FL 1500 (Product # A44115).. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of GM130 was achieved by transfecting HeLa cells with GM130 specific siRNA (Silencer® select Product # s5942) . Western blot analysis (Fig a) was performed using whole cell extracts from GM130 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-GM130 Recombinant Rabbit Monoclonal Antibody (Product # 703794, 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 GM130.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous GM130 using Anti-GM130 Recombinant Rabbit Monoclonal Antibody (Product # 703794, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731, 1:2000). Panel a) shows representative cells that were stained for detection and localization of GM130 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, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating golgi localization of GM130. Panel e) represents control cells with no primary antibody to assess the background. The images were captured at 60X magnification.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of GM130 was achieved by transfecting HeLa cells with specific siRNA (Silencer® select Product # s5942). Immunofluorescence analysis was performed on HeLa cells (untransfected, panel a-d), transfected with GM130 specific siRNA (panel i-l) or non-specific scrambled siRNA (panels e-h). Cells were fixed, permeabilized, and labeled with Anti-GM130 Recombinant Rabbit Monoclonal Antibody (Product # 703794, 1:100 dilution), followed by Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731, 1:2000).. Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) 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 GM130 (green). The images were captured at 60X magnification.