Anti-RhoA antibody (ab86297)

Rabbit polyclonal RhoA antibody. Validated in WB, ICC/IF and tested in Mouse, Rat, Human. Cited in 7 publication(s). Independently reviewed in 1 review(s).

Reviews (1)Q&A (6)References (7)

Overview

  • Product name

    Anti-RhoA antibody
    See all RhoA primary antibodies

  • Description

    Rabbit polyclonal to RhoA

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, WBmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Cow, Dog, Zebrafish, Orangutan

  • Immunogen

    Synthetic peptide corresponding to Human RhoA aa 150 to the C-terminus conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab101307)

  • Positive control

    • This antibody gave a positive signal in the following whole cell lysates: HL60; MCF7; MDA-MB-231; HeLa; Human Platelets.

Properties

Applications

Our Abpromise guarantee covers the use of ab86297 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
ICC/IF Use a concentration of 10 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 20 kDa (predicted molecular weight: 21 kDa).

Target

  • Function

    Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders. May be an activator of PLCE1. Activated by ARHGEF2, which promotes the exchange of GDP for GTP.

  • Sequence similarities

    Belongs to the small GTPase superfamily. Rho family.

  • Domain

    The basic-rich region is essential for yopT recognition and cleavage.

  • Post-translational
    modifications

    Substrate for botulinum ADP-ribosyltransferase.
    Cleaved by yopT protease when the cell is infected by some Yersinia pathogens. This removes the lipid attachment, and leads to its displacement from plasma membrane and to subsequent cytoskeleton cleavage.
    AMPylation at Tyr-34 and Thr-37 are mediated by bacterial enzymes in case of infection by H.somnus and V.parahaemolyticus, respectively. AMPylation occurs in the effector region and leads to inactivation of the GTPase activity by preventing the interaction with downstream effectors, thereby inhibiting actin assembly in infected cells. It is unclear whether some human enzyme mediates AMPylation; FICD has such ability in vitro but additional experiments remain to be done to confirm results in vivo.
    Ubiquitinated by the BCR(BACURD1) and BCR(BACURD2) E3 ubiquitin ligase complexes, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration.

  • Cellular localization

    Cell membrane. Cytoplasm > cytoskeleton.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Aplysia ras related homolog 12 antibody
    • ARH12 antibody
    • ARHA antibody
    • H 12 antibody
    • H12 antibody
    • Oncogene RHO H12 antibody
    • Ras homolog family member A antibody
    • Ras homolog gene family member A antibody
    • Rho A antibody
    • Rho cDNA clone 12 antibody
    • RHO H12 antibody
    • RHO12 antibody
    • RHOA antibody
    • RHOA_HUMAN antibody
    • RHOH12 antibody
    • Small GTP binding protein Rho A antibody
    • Transforming protein Rho A antibody
    • Transforming protein RhoA antibody
    see all

Images

  • Western blot - Anti-RhoA antibody (ab86297)
    Western blot - Anti-RhoA antibody (ab86297)
    All lanes : Anti-RhoA antibody (ab86297) at 1 µg/ml

    Lane 1 : HL60 (Human promyelocytic leukemia cell line) Whole Cell Lysate
    Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 3 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 5 : Human Platelet (Human adult normal cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 21 kDa
    Observed band size: 20 kDa
    why is the actual band size different from the predicted?


    Exposure time: 90 seconds
  • Western blot - Anti-RhoA antibody (ab86297)
    Western blot - Anti-RhoA antibody (ab86297)
    All lanes : Anti-RhoA antibody (ab86297) at 1 µg/ml

    Lane 1 : Kidney (Mouse) Tissue Lysate
    Lane 2 : Mouse lung normal tissue lysate - total protein (ab29297)
    Lane 3 : Kidney (Rat) Tissue Lysate
    Lane 4 : Lung (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 21 kDa
    Observed band size: 20 kDa why is the actual band size different from the predicted?


    Exposure time: 2 minutes
  • Western blot - Anti-RhoA antibody (ab86297)
    Western blot - Anti-RhoA antibody (ab86297)
  • Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody (ab86297)
    Immunocytochemistry/ Immunofluorescence - Anti-RhoA antibody (ab86297)
    ICC/IF image of ab86297 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab86297, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:

  • Abou-Antoun TJ  et al. Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance. PLoS One 13:e0189711 (2018). Read more (PubMed: 29298329) »
  • Ye Y  et al. Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer. Oncotarget 8:94834-94849 (2017). Read more (PubMed: 29212270) »
See all 7 Publications for this product

Publishing research using ab86297? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-RhoA antibody

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (Uterus)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer pH=6.0
Permeabilization
No
Specification
Uterus
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Formaldehyde
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Abcam user community

Verified customer

Submitted Oct 14 2016

Question

Thanks for your reply. I did think the size difference is probably due to interactions or modifications. The modifications I am aware of at the moment I expect to be present in the membrane fraction rather than the cytosolic fraction but I will investigate the literature for clues. In the whole cell fraction I only get the 30 kDa band but if the size difference is due to differential properties of membrane and cytosolic RhoA the lack of the smaller band could be explained by RhoA being more dilute in the whole cell fraction than in the membrane. I get my whole cell fraction as part of the fractionation protocol in which I unfortunately can’t use RIPA buffer but I have already added a step to my protocol that will hopefully improve solubilisation if it is the problem. And if this doesn’t help I will try heating with and without SDS and DTT.

Thanks for your help.

Regards,

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Answer

Thank you for your reply.

I hope the use of SDS and DTT will resolve the problem!

Please let us know also if there is anything else we could help you with. We do have for example different cell fractionation kits, and if the SDS and DTT would not clarify the band pattern, we could try sucha kit to see whether the same bands are observed in the different fractions (I problably could make a good testing offer on these if you would then provide a feedback as well).

I wish you all the best for your project and please do not hesitate to contact us again for any questions in relation to our products.

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Question

Sorry it’s taken me such a long time to get back to you. I did manage to get the RhoA antibody working though something slightly unexpected happened and I’m hoping you might be able to help me with this. I am doing subcellular fractionation and depending on the fraction the bands were different sizes; in the nuclear and membrane fractions the bands were about the expected size (or maybe slightly smaller) but in the whole cell and cytosolic fractions the bands were about 30 kDa. The only difference I can think of is the presence of detergent; the nuclear and membrane fractions both have it and not the cytosolic, I have added detergent to the whole cell fraction as well though differently from the nuclear and membrane fractions. Any more info on this would be useful.



Regards,

Read More
Answer

Thank you very much for your email. I am very happy to hear that the RhoA antibody is working now.

The observedband in the cytoplasm is indeed unexpected. I would think this would correspond to a complex in the cytoplasm or another modification.I am however not an expert in RhoA biology and would rely ona more thorough literature research for finding what interaction partner it could be.

In order to analyse howeverfroma technical point of viewthis observed protein, I would recommend to systematically heat with and without SDS and beta Mercaptoethanol (or DTT) the fractions, and see whether the bands are still present. Do you observe the 20kD band also in the whole cell fraction?

What type of buffer have you used to make the cell lysis? I can recommend also to try the RIPA buffer for the whole cell lysate. RIPA buffer is a very good buffer to solubilize the proteins. (https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1)

Please let me know if this makes sense. I hope this will help to clarify the results. Please keep me updated and do let me know if you would have any doubt about the quality of the antibody.

I am looking forward to hear back from you.

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Question

What is the homology between the immunogen and the mouse RhoB and RhoC protein sequences?

Read More
Answer

Thank you for your call a couple weeks ago, and I sincerely apologize for the delay in getting back to you.
As we discussed, the immunogen of ab86297 is 83% identical to RhoB and 73% to RhoC, so it is possible that this antibody will cross-react. I have unfortunately been unable to obtain any further information about the immunogen of ab68826.

I am sorry that I can not be more help in this situation, but please let me know if there is anything else that I might be able to do for you.

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Question

Thanks for your reply. Yes the samples have been reducedand denaturedand I know there is protein there asb-actin comes up clearly. Also I have used protease inhibitors. I have used the secondary antibody withanotherrabbit primary and that works fine.Ihaven't tried BSA for blocking yet but will do that as soon as possible.

Thanks,

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Answer

Thank you for your email.

I look forward hearing back from you with the results of the BSA blocking.

Have a great day!

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Question

Inquiry: I am not able to get signal from this antibody. I have been testing it in rat liver and have used 20-40 ug of protein. I generally use 4-12% gel as this should be fine for the protein size though I have tried a higher percentage gel as well. Transfers I usually run at 75V for 1.5 hours. I block for 1 hour in room temperature in 5% milk. Primary antibody incubation I do overnight at 4C and I have tried 1:1000 and 1:750 dilutions in milk. Secondary antibody I use at 1:100 000 in RT for 1.5 hours which I know works with another antibody. Washes I do 3 times 7-10 minutes.

Read More
Answer

Thank you for taking time to contact us with this information. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab86297. I would also appreciate if you can confirm some further details:

1.) Can you please confirm the samples have been reduced and denatured? Reducing with DTT or beta Mercaptoethanol and denaturing the samples with SDS and heating 5 to 10 minutes at 95°C will help the proteins to enter the gel and resolvenicely.

2.) I can strongly recommend to use BSA as a blocking buffer. Indeed, the laboratory has tested this antibody using BSAfor blocking. Changing the blocking buffer can significantly improve the results. As an illustration, please see also the Western blot image on the datasheet of ab9385 https://www.abcam.com/index.html?datasheet=9385 (or use the following: https://www.abcam.com/index.html?datasheet=9385).

3.) Can you also confirm, whether the other reagents can be exluded to be at the origin of the problem? - Has the secondary antibody already provided good results with other primary rabbit antibodies?

4.) In order to exclude protein degradation as possible origin of the problem, can you please confirm whether proteases inhibitors have been added to the lysate and whetherother proteins been detected in the same lysates?

I agree also that liver cells should have a high level of RhoA. Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Question

Will this antibody also react with RhoB or RhoC?

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Answer

Thank you for contacting Abcam regarding ab86297. We have not tested this antibody for cross reactivity with RhoB or RhoC.  By blasting the immunogen sequence for this Ab, it shows there may be some cross reactivity with RhoC. I hope this information is helpful.  Please do not hesitate to contact us if you have any additional questions.

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