Antibodies, Proteins, Kits and Reagents for Life Science

338 390 ₸ Product size

100 µl 338 390 ₸

Order now and get it on Tuesday March 02, 2021

Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EP786Y] to ROCK2 + ROCK1
Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt
Reacts with: Mouse, Rat, Human

Product name

Anti-ROCK2 + ROCK1 antibody [EP786Y]
Description

Rabbit monoclonal [EP786Y] to ROCK2 + ROCK1
Host species

Rabbit
Specificity

This antibody recognizes both the cleaved C-terminus of ROCK 1 (30 kDa) and full length protein (158 kDa). The immunogen used for this product shares 83% homology with ROCK2 and has been shown to bind recombinant human ROCK2, please see western blot images below.

Tested Applications & Species

Application	Species
Flow Cyt	Human
ICC/IF	Human
IHC-P	Human
IP	Human
WB	Rat Human

See all applications and species data

Immunogen

Synthetic peptide within Human ROCK1 aa 1100-1200 (C terminal). The exact sequence is proprietary.
Database link: Q13464
Positive control
- WB: Untreated, Calyculin A treated and Camptothecin treated HeLa whole cell lysate (ab150035). Jurkat, Ramos, PC-12 and RAW264.7 cell lysates. IHC-P: Human adenocarcinoma of the colon and thyroid gland carcinoma tissues. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate (ab150035).
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EP786Y
Isotype

IgG
Research areas

Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

All lanes : Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/5000 dilution (purified)

Lane 1 : HeLa cell lysate - treated with Calyculin A
Lane 2 : HeLa cell lysate - treated with Camptothecin
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate
Lane 5 : Ramos cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 158 kDa
Observed band size: 158 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarcinoma of the colon tissue labelling ROCK2 + ROCK1 with purified ab45171 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
Immunocytochemistry/ Immunofluorescence - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ROCK2 + ROCK1 with purified ab45171 at 1/50. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150078, an Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/50) and secondary antibody, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).
Flow Cytometry - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Overlay histogram showing HeLa cells stained with unpurified ab45171 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45171, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

All lanes : Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/1000 dilution

Lane 1 : Recombinant Human ROCK1 protein (aa 1114 to 1354) (30 kDa)
Lane 2 : Recombinant Human ROCK2 protein (aa 1132 to 1388) (30 kDa)

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 158 kDa
Observed band size: 30 kDa
why is the actual band size different from the predicted?

Loading control: Anti-6X His tag® antibody [EPR20547] (ab213204)

Blocking buffer and concentration: 5% NFDM/TBST

Exposure Times:

Lane 1: 3 seconds

Lane 2: 7 seconds
Immunoprecipitation - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

ab45171 (purified) at 1/40 immunoprecipitating ROCK2 + ROCK1 in HeLa cell lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.
Western blot - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

All lanes : Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/5000 dilution (purified)

Lane 1 : PC-12 cell lysate
Lane 2 : RAW264.7 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 158 kDa
Observed band size: 158 kDa

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) This image is courtesy of an anonymous Abreview

Unpurified ab45171 staining ROCK2 + ROCK1 in mouse embyro 18dpc tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 2 hours at room temperature; antigen retrieval was by heat mediation in Tris pH 9. Samples were incubated with primary antibody (1/100 in 1% BSA + 1% FBS in TBS) for 16 hours. An undiluted HRP-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.
See Abreview
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid gland carcinoma tissue labelling ROCK2 + ROCK1 with unpurified ab45171.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171)

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