Recombinant
RabMAb

Recombinant Anti-ROCK1 antibody [EP786Y] (ab45171)

Reviews (7)Q&A (11)References (57)

Overview

  • Product name

    Anti-ROCK1 antibody [EP786Y]
    See all ROCK1 primary antibodies

  • Description

    Rabbit monoclonal [EP786Y] to 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. Cross-reactivity with this protein has not been confirmed experimentally.

  • Tested applications

    Suitable for: IHC-Fr, WB, IHC-P, ICC/IF, IP, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human

  • 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.

Properties

Applications

Our Abpromise guarantee covers the use of ab45171 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr 1/100. PubMed: 19295659
WB 1/2000 - 1/10000. Predicted molecular weight: 158 kDa.

For unpurified use at 1/500.
IHC-P 1/50 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.
ICC/IF 1/50 - 1/500.

 
IP 1/40 - 1/50.

 
Flow Cyt 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function

    Protein kinase which is a key regulator of actin cytoskeleton and cell polarity. Involved in regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of DAPK3, GFAP, LIMK1, LIMK2, MYL9/MLC2, PFN1 and PPP1R12A. Phosphorylates FHOD1 and acts synergistically with it to promote SRC-dependent non-apoptotic plasma membrane blebbing. Phosphorylates JIP3 and regulates the recruitment of JNK to JIP3 upon UVB-induced stress. Acts as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability. Acts as a negative regulator of VEGF-induced angiogenic endothelial cell activation. Required for centrosome positioning and centrosome-dependent exit from mitosis. Plays a role in terminal erythroid differentiation. May regulate closure of the eyelids and ventral body wall by inducing the assembly of actomyosin bundles. Promotes keratinocyte terminal differentiation. Involved in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process, essential for osteoblast mineralization.

  • Tissue specificity

    Detected in blood platelets.

  • Sequence similarities

    Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family.
    Contains 1 AGC-kinase C-terminal domain.
    Contains 1 PH domain.
    Contains 1 phorbol-ester/DAG-type zinc finger.
    Contains 1 protein kinase domain.
    Contains 1 REM (Hr1) repeat.

  • Domain

    The C-terminal auto-inhibitory domain interferes with kinase activity. RHOA binding leads to a conformation change and activation of the kinase. Truncated ROCK1 is constitutively activated.

  • Post-translational
    modifications

    Autophosphorylated on serine and threonine residues.
    Cleaved by caspase-3 during apoptosis. This leads to constitutive activation of the kinase and membrane blebbing.

  • Cellular localization

    Cytoplasm. Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole. Golgi apparatus membrane. Cell projection, bleb. Cytoplasm, cytoskeleton. Cell membrane. Cell projection, lamellipodium. Cell projection, ruffle. Associated with the mother centriole and an intercentriolar linker. Colocalizes with ITGB1BP1 and ITGB1 at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. Localizes at the cell membrane in an ITGB1BP1-dependent manner (By similarity). A small proportion is associated with Golgi membranes.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • coiled-coil-containing protein kinase 1 antibody
    • coiled-coil-containing protein kinase I antibody
    • MGC131603 antibody
    • MGC43611 antibody
    • p160 Rhoassociated coiled coil-forming protein kinase antibody
    • p160 ROCK-1 antibody
    • p160 ROCK1 antibody
    • p160ROCK antibody
    • PRO0435 antibody
    • Renal carcinoma antigen NY REN 35 antibody
    • Renal carcinoma antigen NY-REN-35 antibody
    • Rho associated coiled coil containing protein kinase 1 antibody
    • Rho associated protein kinase 1 antibody
    • Rho kinase antibody
    • Rho-alpha kinase antibody
    • Rho-associated antibody
    • Rho-associated protein kinase 1 antibody
    • ROCK I antibody
    • ROCK-I antibody
    • ROCK1 antibody
    • ROCK1_HUMAN antibody
    • Rok antibody
    • rokalpha antibody
    see all

Images

  • Western blot - Anti-ROCK1 antibody [EP786Y] (ab45171)
    Western blot - Anti-ROCK1 antibody [EP786Y] (ab45171)
    All lanes : Anti-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-ROCK1 antibody [EP786Y] (ab45171)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK1 antibody [EP786Y] (ab45171)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarcinoma of the colon tissue labelling 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-ROCK1 antibody [EP786Y] (ab45171)
    Immunocytochemistry/ Immunofluorescence - Anti-ROCK1 antibody [EP786Y] (ab45171)

    Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling 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-ROCK1 antibody [EP786Y] (ab45171)
    Flow Cytometry - Anti-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.

  • Immunoprecipitation - Anti-ROCK1 antibody [EP786Y] (ab45171)
    Immunoprecipitation - Anti-ROCK1 antibody [EP786Y] (ab45171)

    ab45171 (purified) at 1/40 immunoprecipitating 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-ROCK1 antibody [EP786Y] (ab45171)
    Western blot - Anti-ROCK1 antibody [EP786Y] (ab45171)
    All lanes : Anti-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-ROCK1 antibody [EP786Y] (ab45171)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK1 antibody [EP786Y] (ab45171)This image is courtesy of an anonymous Abreview

    Unpurified ab45171 staining 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-ROCK1 antibody [EP786Y] (ab45171)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROCK1 antibody [EP786Y] (ab45171)

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

References

This product has been referenced in:

  • Yang Z  et al. DLC2 operates as a tumor suppressor gene in breast cancer via the RhoGTPase pathway. Oncol Lett 17:2107-2116 (2019). Read more (PubMed: 30719106) »
  • Deng R  et al. lncRNA SNHG1 negatively regulates miRNA-101-3p to enhance the expression of ROCK1 and promote cell proliferation, migration and invasion in osteosarcoma. Int J Mol Med 43:1157-1166 (2019). Read more (PubMed: 30592267) »
See all 57 Publications for this product

Publishing research using ab45171? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 18 Abreviews or Q&A

IHC - Wholemount abreview for Anti-ROCK1 antibody [EP786Y]

 Excellent
Abreviews
abreview image
Application
IHC - Wholemount
Sample
Mouse Embryo (Apical neuroepithelium)
Specification
Apical neuroepithelium
Read More

Dr. Gabriel Galea

Verified customer

Submitted Jan 21 2020

Western blot abreview for Anti-ROCK1 antibody [EP786Y]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Pig Cell lysate - whole cell (heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
5 µg
Specification
heart
Blocking step
Licor as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 24°C
Read More

Abcam user community

Verified customer

Submitted Nov 04 2016

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-ROCK1 antibody [EP786Y]

 Good
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
FBS / BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris pH 9
Sample
Human Tissue sections (Infantile fibromatosis)
Specification
Infantile fibromatosis
Permeabilization
No
Fixative
Formaldehyde
Read More

Abcam user community

Verified customer

Submitted Jul 04 2014

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-ROCK1 antibody [EP786Y]

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
FBS / BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris pH 9
Sample
Mouse Tissue sections (Mouse embryo 18dpc)
Specification
Mouse embryo 18dpc
Permeabilization
No
Fixative
Formaldehyde
Read More

Abcam user community

Verified customer

Submitted Jan 15 2014

Western blot abreview for Anti-ROCK1 antibody [EP786Y]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF)
Loading amount
30 µg
Specification
MEF
Treatment
TGF b1 o/n 5 ug/ul
Gel Running Conditions
Reduced Denaturing (7.5)
Blocking step
(agent) for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Abcam user community

Verified customer

Submitted Feb 27 2013

Question

Hi I understand, so can we try the different antibody ab143181 please.



Thanks

Read More
Answer

Thank you for confirming these details and for your cooperation.

As requested, I have issued a free of charge replacement of alternative ab134181 Anti-ROCK1 antibody [EPR638Y]with the order number #####.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to letme know. I sincerely hope this vial will work in your experiments. If not, please do not hesitate to contact me and I suggest in this case the best way to proceed would be to provide a refund or credit note for the original antibody.

I wish you the best of luck and look forward to hearing from you with the results of the experiments with the new antibody.

Read More

Question

Hi following on from the email correspondence below, can you let me know if you have a new parent lot yet for ab45171? I really need to get the antibody working again. If not can I try an aliquot of ab134181 which appears to be a new ROCK1 antibody

Thanks

Read More
Answer

Thank you once again for your message. Thank you also for your patience waiting for a response, I wanted to ensure I had time to review the whole case again fully before contacting you as it has been a while since we last corresponded.

I am sorry to confirm that we still only havethe same parent lot ofab45171ROCK1 antibody [EP786Y]available. I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Many customers are purchasing this product and when I checked the records,we have received no other complaints in the last year for this product. It is unusual and regrettable that several vials have not worked for you.

I would be pleased to provide a replacement from the same lot, which will still be covered by our guarantee. Alternatively, I fully understand your concerns and you prefer a credit note in this case, or an alternative replacement of ab134181I will be pleased to arrange this for you.

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

Read More

Question

Hi we have tested the new ROCK1 antibody ab97592 using the same conditions/method we always used for ab45171 when it was working, but unfortunately the new antibody gave just non-specific staining. I was wondering if it was possible to try ab58305 as an alternative to see if this one will work?

Thanks

Read More
Answer

Thank you for your message. I am sorry to hear that the alternative free of charge replacement has also not worked.

It is regrettable both the antibodies have not provided satisfactory results. I would like to reassure you that ab97592 is tested and guaranteed for IHC-P on human samples.

I am concerned that these experiments are still not working for you. In this case, I would like to investigate further Before deciding how to proceed in this case I would appreciate if you are able to confirm the following information which may help me to determine where the difficulty is:

1. Please confirm what the species and tissue typeof the sample. Are these frozen (IHC-Fr) or paraffin embedded sections (IHC-P)?

2. Please confirm how the samples are being processed. Are the samples fresh before fixation? How are they being fixed?

3. What dilution of antibody is being used? Has this been optimized?

4. How is the antibody being stored?

5. Please confirm the wash steps being used?

6. Is the current vial of secondary antibody working well with other primary antibodies? Please confirm the details for the primary antibody. Has a no primary control been included?

Many thanks for your continued cooperation. I look forward to hearing from you with the requested information.

Read More

Question

I think I wrote it in the wrong way,
I would like to test an antibody that can detect ROCK (PCP pathway) in westerns and immuno staining. I does not need to inhibit the signalling itself.
Which one can you recommend?

Read More
Answer

Thank you for contacting us.

All the antibodies are well characterized, please choose based on your requirements. e.g polyclonal/ monoclonal, Rabbit/mouse/sheep etc.

The catalogue numbers are:

ab13660, ab45171, ab56661, ab58305, ab125025

ab71598, ab66310, ab10549, ab97592, ab85612

When selecting check the Abreview and choose the antibody which have more Abreview and publications.

You may also interested in testing the antibody. Please click the below link for more info.

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

I have been using some of antibodies a lot for Immuno-Staining and Western Blotting.
At the moment I am interested in looking at a functional ROCK antibody and its regulation during PCP / Wnt signalling.
Is there an antibody in your product catalogue you can recommend ?
Thanks for your help,
Constanze

Read More
Answer

Thank you for contacting us.

We unfortunately do not have anti ROCK antibodies available that can be used for functional assays e.g. activation or inhibition of ROCK. I would suggest searching the similar antibody clone in publications. We have 6-7 monoclonal antibodies in catalogue however we do not know whether these are being used in any functional assay. I am sorry we are unable to help with this particular ab.

I hope this information is nevertheless helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

1-10 of 18 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com