Product Uses Include
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.
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)
|Leguay, Kévin et al.||Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry||Journal of Cell Biology||2022||ISSN 1540-8140|
|Khan, Alamzeb et al.||ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2022||ISSN 1530-6860|
|You, Jae Sung et al.||ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through Autophagy||Cell Reports||2021||ISSN 2211-1247|
|Afanasyeva, Elena A. et al.||Kalirin-RAC controls nucleokinetic migration in ADRN-type neuroblastoma||Life Science Alliance||2021||ISSN 2575-1077|
|Liang, Xiaoting et al.||Acetylation dependent functions of Rab22a-NeoF1 Fusion Protein in Osteosarcoma||Theranostics||2020||ISSN 1838-7640|
|Addis, Dylan R. et al.||Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure||American Journal of Physiology - Lung Cellular and Molecular Physiology||2020||ISSN 1522-1504|
|Evans, Frances et al.||Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells||Journal of Cellular Physiology||2020||ISSN 1097-4652|
|Huang, Yuxing et al.||Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundance||Cells||2019||ISSN 2073-4409|
|Choi, Michael Y. et al.||Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia||Cell Stem Cell||2018||ISSN 1875-9777|
|Yu, Lixia et al.||C-Maf inducing protein inhibits coflin-1 activity and alters podocyte cytoskeleton organization||Molecular Medicine Reports||2017||ISSN 1791-3004|
|Kempf, Anissa et al.||Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG Interaction||Developmental Cell||2017||ISSN 1878-1551|
|Li, Shufeng et al.||Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth||Biomaterials||2015||ISSN 1878-5905|
|Gray, Jason D. et al.||LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure||Human Molecular Genetics||2013||ISSN 0964-6906|
|Xu, Jie et al.||RhoGAPs Attenuate Cell Proliferation by Direct Interaction with p53 Tetramerization Domain||Cell Reports||2013||ISSN 2211-1247|
|Buranda, Tione et al.||Rapid parallel flow cytometry assays of active GTPases using effector beads||Analytical Biochemistry||2013||ISSN 1096-0309|
|Sabbatini, Maria Eugenia et al.||Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling||PLoS ONE||2013||ISSN 1932-6203|
|Zhang, Y. et al.||High glucose-induced RhoA activation requires caveolae and PKCβ1-mediated ROS generation||American journal of physiology. Renal physiology||2012||ISSN 1522--1466|
|Ponsaerts, Raf et al.||RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system||PLoS ONE||2012||ISSN 1932-6203|
|Cartier-Michaud, Amandine et al.||Matrix-bound PAI-1 supports cell blebbing via RhoA/rock1 signaling||PLoS ONE||2012||ISSN 1932-6203|
|Kyrkou, A. et al.||RhoD participates in the regulation of cell-cycle progression and centrosome duplication||Oncogene 2013 32:14||2012||ISSN 1476--5594|
|Ladhani, Omar et al.||Pigment Epithelium-Derived Factor Blocks Tumor Extravasation by Suppressing Amoeboid Morphology and Mesenchymal Proteolysis||Neoplasia (New York, N.Y.)||2011||ISSN 1476-5586|
|Chacon, Pedro J. et al.||Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid β toxicity||Molecular Neurodegeneration||2011||ISSN 1750-1326|
|Bi, Yan et al.||A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini||American journal of physiology. Cell physiology||2005||ISSN 0363--6143|
|Ishii, Satoshi et al.||Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor||The Journal of biological chemistry||2005||ISSN 0021--9258|
|Wang, Shizhen Emily et al.||Transforming Growth Factor β (TGF-β)-Smad Target Gene Protein Tyrosine Phosphatase Receptor Type Kappa Is Required for TGF-β Function||Molecular and Cellular Biology||2005||ISSN 0270--7306|
|Saito, Shin'ichi et al.||Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation||The Journal of biological chemistry||2004||ISSN 0021--9258|
|Wang, Qin et al.||Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells||The Journal of biological chemistry||2004||ISSN 0021--9258|
|Zhang, Xiao Feng et al.||Rho-Dependent Contractile Responses in the Neuronal Growth Cone Are Independent of Classical Peripheral Retrograde Actin Flow||Neuron||2003||ISSN 0896-6273|
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 email@example.com.