The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state. Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation. The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors RhoA protein activation. The assay uses the Rho binding domain (RBD) of the Rho effector protein, Rhotekin. The RBD motif has been shown to bind specifically to the GTP-bound form of RhoA. The fact that the RBD region of Rhotekin has a high affinity for GTP-RhoA and that Rhotekin binding results in a significantly reduced intrinsic and catalytic rate of GTP hydrolysis make it an ideal tool for affinity purification of GTP-RhoA from cell lysates. The Rhotekin-RBD protein supplied in this kit contains Rhotekin residues 7-89 and is in the form of a GST fusion protein, which allows one to "pull-down" the Rhotekin-RBD/Rho-GTP complex with brightly colored glutathione affinity beads. The assay therefore provides a simple means of quantitating RhoA activation in cells. The amount of activated RhoA is determined by a western blot using a RhoA specific antibody.
The kit contains sufficient materials for 80 assays depending on activation levels of Rho in cells and includes reagents for positive and negative controls. The following components are included:
Figure 1. The brightly colored glutathione agarose beads in BK036 makes the kit easy to use.
The RhoA activation assay was tested by loading the RhoA protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded RhoA is very efficiently precipitated while very little GDP-loaded RhoA is precipitated (Fig. 2).
Figure 2. Results from BK036 RhoA activation assay. Activated Rho was precipitated and detected in a Western blot using kit BK036. The first lane shows a 50 ng recombinant His-tagged RhoA standard (Rec. His-RhoA). The following lanes shows the pull-down of inactive, GDP-loaded RhoA (RhoA-GDP PD) or active, GTPγS-loaded RhoA (RhoA-GTP PD) from equal amounts of cell lysates.
Please check out the new version of the Rac Activation Assay and associated products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)
C. Yan et al., 2014. Discovery and characterization of small molecules that target the GTPase Ral. Nature. doi: 10.1038/nature13713.
Xie et al., 2013. Activation of RhoA/ROCK regulates NF-κB signaling pathway in experimental diabetic nephropathy. Mol. Cell. Endocrinol. 369, 86-97.
Zhan et al., 2013. The effect of an NgR1 antagonist on the neuroprotection of cortical axons after cortical infarction in rats. Neurochem. Res. doi: 10.1007/s11064-013-1026-z.
Tong et al., 2013. Activation of RhoA in alcohol-induced intestinal barrier dysfunction. Inflammation. doi: 10.1007/s10753-013-9601-7.
Cheng et al., 2012. Infrasonic noise induces axonal degeneration of cultured neurons via a Ca2+ influx pathway. Toxicol. Lett. v 212, pp 190-197.
Lee et al., 2012. Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol. Appl. Pharmacol. v 262, pp 91-98.
Antonyak et al., 2012. RhoA triggers a specific signaling pathway that generates transforming microvesicles in cancer cells. Oncogene. doi:10.1038/onc.2011.636.
Nithipatikom et al., 2012. Cannabinoid receptor type 1 (CB1) activation inhibits small GTPase RhoA activity and regulates motility of prostate carcinoma cells. Endocrinology. v 153, pp 29-41.
Droppelmann et al., 2012. Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis. Neurobiol. Aging. http://dx.doi.org/10.1016/j.neurobiolaging.2012.06.021.
Otani et al., 2011. Involvement of protein kinase C and RhoA in protease-activated receptor 1–mediated f-actin reorganization and cell growth in rat cardiomyocytes. J. Pharmacol. Sci. v 115, pp 135-143.
Khan et al., 2011. Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. J. Clin. Invest.doi:10.1172/JCI43758.
Yi et al., 2010. Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the Rho A-Rho-kinase pathway in rats. J. Cardiovasc. Pharmacol. v 57, pp 231-239.
Pixley et al., 2005. BCL6 suppresses RhoA activity to alter macrophage morphology and motility. J. Cell Sci. v 118, pp 1873-1883.
Birukova et al., 2004. Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am. J. Physiol. v 287, pp L86-L93.
Cetin et al., 2004. Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J. Biol. Chem. v 279, pp 24592-24600.
Orr et al., 2004. Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J. Biol. Chem. v 279, pp 48983-48992.
Sasai et al., 2004. The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. v 6, pp 741-748.
Setiadi and McEver, 2003. Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow. J. Cell Biol. v 163, pp 1385-1395.
Question 1: I have high background and/or multiple bands on my western blot. How can I fix this?
Answer 1: There are multiple causes of high background and/or multiple bands. Some suggestions to improve background signal include:
Question 2: How much of the beads should I use for my pull-down experiments?
Answer 2: Rhotekin-RBD 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 RhoA 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 3: How can I test whether the beads are working properly?
Answer 3: A standard biological assay for rhotekin-RBD beads consists of a RhoA protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # RT02 and BK036 datasheets 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.
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