LF-PA0049

antibody from Invitrogen Antibodies

Targeting: ROCK2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Data presented on provider website)

Immunohistochemistry (Recommended by provider)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

LF-PA0049

Provider

Invitrogen Antibodies

Product name

Anti-ROCK2 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

A suggested positive control for this product is HepG2 cells.

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

Lot Dependent

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of ROCK2 was achieved by transfecting HeLa with ROCK2 specific siRNAs (Silencer® select Product # s18162). Western blot analysis (Fig. a) was performed using whole cell extracts (1% SDS) from the ROCK2 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ROCK2 Polyclonal Antibody (Product # LF-PA0049, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 ROCK2.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-ROCK2 Polyclonal Antibody (Product # LF-PA0049) and a 180 kDa corresponding to ROCK2 was observed across cell lines and tissue extract tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), MCF-7 (Lane 3), MDA-MB-231 (Lane 4), U-87 MG (Lane 5), C1C12 (Lane 6) and Rat Brain (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of ROCK2 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 ROCK2 Rabbit Polyclonal Antibody (Product # LF-PA0049) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing Cytoplasmic and membrane localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.