Rhotekin-RBD beads (binds active Rho proteins)

Rhotekin-RBD beads (binds active Rho proteins)

Product Uses Include

  • Measurement of the GTP/GDP ratio of RhoA/B/C in vitro.
  • Quantitation of GTP-RhoA/B/C from tissue and tissue culture cell lysates.

The Rho binding domain (RBD) of the human Rhotekin protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, RhoA, RhoB and RhoC proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Rho proteins as well as to specifically block the activity of these proteins in vitro and in vivo.

The GST-Rhotekin-RBD contains residues 7-89 of Rhotekin. This region includes the sequences required for the high affinity interaction with GTP-Rho.

The protein is supplied in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. This product is used in our RhoA pulldown activation assay Biochem Kit™ (Cat. # BK036). The GST-Rhotekin-RBD is also available as a free protein (Cat. # RT01).


Figure 1:  GST-Rhotekin-RBD protein purity determination. A 10 µg sample of PAK01 was  

Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Rhotekin-RBD protein is ~85% pure (see Figure 2).


Figure 2: GST-Rhotekin-RBD protein purity determination. A 20 µg sample of RT02 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-Rhotekin-RBD protein runs at approximately 35 kDa. 

Biological Activity
The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound, form of Rho protein. It has a much lower affinity for the inactive, GDP-bound, form of Rho. Biological activity of Rhotekin-RBD protein is therefore determined by its selectivity for GTP-Rho protein. The assay for monitoring the GTP-bound form of Rho is a protein pulldown assay using RT02 beads and a human platelet extract loaded with GTPγS (Cat. # BS01)

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Morel, Anne et al.Bone resorption by osteoclasts involves fine tuning of RHOA activity by its microtubule-associated exchange factor GEF-H1Frontiers in Physiology2024ISSN 1664-042X
Ren, Xiaoyu et al.Loss of Myo19 increases metastasis by enhancing microenvironmental ROS gradient and chemotaxisEMBO Reports2024ISSN 1469--3178
Samain, Remi et al.CD73 controls Myosin II–driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cellsScience Advances2023ISSN 2375-2548
Wong, Darren Chen Pei et al.Hiltonol, a dsRNA Mimic, Promotes NK Cell Anticancer Cytotoxicity Through TAZ Cytoplasmic SequestrationAdvanced Therapeutics2023ISSN 2366--3987
Zhang, Xingyu et al.Soluble TREM2 ameliorates tau phosphorylation and cognitive deficits through activating transgelin-2 in Alzheimer’s diseaseNature Communications 2023 14:12023ISSN 2041--1723
Morishita, Jun et al.Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cellsOpen Biology2023ISSN 2046-2441
Leguay, Kévin et al.Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entryJournal of Cell Biology2022ISSN 1540-8140
Khan, Alamzeb et al.ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leakFASEB journal : official publication of the Federation of American Societies for Experimental Biology2022ISSN 1530-6860
You, Jae Sung et al.ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through AutophagyCell Reports2021ISSN 2211-1247
Afanasyeva, Elena A. et al.Kalirin-RAC controls nucleokinetic migration in ADRN-type neuroblastomaLife Science Alliance2021ISSN 2575-1077
Evans, Frances et al.Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cellsJournal of Cellular Physiology2020ISSN 1097-4652
Addis, Dylan R. et al.Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposureAmerican Journal of Physiology - Lung Cellular and Molecular Physiology2020ISSN 1522-1504
Liang, Xiaoting et al.Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in OsteosarcomaTheranostics2020ISSN 1838-7640
Huang, Yuxing et al.Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundanceCells2019ISSN 2073-4409
Choi, Michael Y. et al.Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic LeukemiaCell Stem Cell2018ISSN 1875-9777
Yu, Lixia et al.C-Maf inducing protein inhibits coflin-1 activity and alters podocyte cytoskeleton organizationMolecular Medicine Reports2017ISSN 1791-3004
Kempf, Anissa et al.Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG InteractionDevelopmental Cell2017ISSN 1878-1551
Li, Shufeng et al.Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growthBiomaterials2015ISSN 1878-5905
Sabbatini, Maria Eugenia et al.Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 SignalingPLoS ONE2013ISSN 1932-6203
Gray, Jason D. et al.LRP6 exerts non-canonical effects on Wnt signaling during neural tube closureHuman Molecular Genetics2013ISSN 0964-6906
Xu, Jie et al.RhoGAPs Attenuate Cell Proliferation by Direct Interaction with p53 Tetramerization DomainCell Reports2013ISSN 2211-1247
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Question 1:  How much of the beads should I use for my pull-down experiments?

Answer 1:  Rhotekin-RBD protein GST beads (Cat. # RT02) will bind to Rho-GDP with a much lower affinity than Rho-GTP.  If too many Rhotekin-RBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) RhoA.  The result of this will be an underestimation of RhoA activation.  For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Rho activation or inactivation assay.  Once optimal conditions have been established, bead titrations should no longer be necessary.  We recommend 25, 50 and 100 μg bead titrations.


Question 2:  How can I test whether the beads are working properly?

Answer 2:  A standard biological assay for Rhotekin-RBD protein GST beads consists of a Rho protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP.  Here are guidelines to follow (see RT02 datasheet for more details):

Positive Cellular Protein Control:

Total cell lysate (300 – 800 μg) should be loaded with GTPγS as a positive control for the pull-down assay.  The following reaction details how to load endogenous RhoA with the nonhydrolysable GTP analog (GTPγS).  This is an excellent substrate for Rhotekin-RBD beads and should result in a strong positive signal in a pull-down assay.

 a) Perform GTP loading on 300 – 800 μg of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTPγS (200 μM final concentration). Under these conditions 5 - 10% of the RhoA protein will load with non-hydrolysable GTPγS and will be “pulled down” with the Rhotekin-RBD beads in the assay.

c) Incubate the control sample at 30°C for 15 min with gentle rotation.

d) Stop the reaction by transferring the tube to 4°C and adding 1/10th volume of STOP Buffer.

e) Use this sample immediately in a pull-down assay.

 Negative Cellular Protein Control:

This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTPγS.  Loading endogenous RhoA with GDP will inactivate RhoA and this complex will bind very poorly to Rhotekin-RBD beads.


 If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.