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Rhotekin RBD protein on GST beads

Rhotekin RBD protein on GST beads

ArtNr	RT02-A
Hersteller	Cytoskeleton
Menge	2 x 2 mg
Quantity options	2 x 2 mg 6 x 2 mg
Kategorie	Oncology Neuroscience Neural Lineage Markers Infectious Diseases Viral Infections Coronavirus (COVID-19) Cell Biology Cytoskeleton Reagents & Chemicals Nanoparticles Beads & Resin By Organ Testicular & Prostate Cancer Pancreatic Cancer Neurodegeneration Markers small GTPases Rho Subfamily
Typ	Proteins
Specific against	other
Purity	Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Rhotekin-RBD protein is ca.85% pure (see Figure 2)
Citations	Kyrkou et al., 2012. RhoD participates in the regulation of cell-cycle progression and centrosome duplication. Oncogene. doi:10.1038/onc.2012.195. Zhang et al., 2012. High glucose-induced RhoA activation requires caveolae and PKC&beta,1-mediated ROS generation. AJP - Renal Physiol. v 302, pp F159-F172. Chacon et al., 2011. Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid b toxicity. Mol. Neurodegen. v 6, p 14. Ladhani et al., 2011. Pigment Epithelium&ndash,Derived Factor Blocks Tumor Extravasation by Suppressing Amoeboid Morphology and Mesenchymal Proteolysis. Neoplasia. v 13, pp 633&ndash,642. Papadimitriou et al., 2011. TGF&beta,-induced Early Activation of the Small GTPase RhoA is Smad2/3-independent and Involves Src and the Guanine Nucleotide Exchange Factor Vav2. Cell Physiol. Biochem. v 28, pp 229-238. Bi and Williams, 2005. A role for Rho and Rac in secretagogue induced amylase release by pancreatic acini. Am. J. Physiol. v 289, pp C22-C33 Ishii et al., 2005. Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J. Biol. Chem. v 280, pp 9083-9087. Wang et al., 2005. Transforming growth factor &beta, (TGF-&beta,)-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-&beta, function. Mol. Cell. Biol. v 25, pp 4703-4715. Saito et al., 2004. Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation. J. Biol. Chem. v 279, pp 7169-7179. Wang et al., 2004. Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells. J. Biol. Chem. v 279, pp 28831-28834. Zhang et al., 2003. Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron. v 40, pp 931-944.
ECLASS 10.1	32160409
ECLASS 11.0	32160409
UNSPSC	12352202
Alias	Small G protein, Rhotekin-RBD protein, Rho binding domain, RBD
Similar products	Small G protein, RBD, Rhotekin-RBD protein, Rho binding domain
Versandbedingung	Raumtemperatur
Lieferbar
Shipping Temperature	AT
Storage Conditions	On Arrival: 4°C
Delivery Time	1-2 Weeks
FAQs	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 &mu, g 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 GTP&gamma, S (Cat. #BS01) or GDP. Here are guidelines to follow (see RT02 datasheet for more details): Positive Cellular Protein Control: Total cell lysate (300 &ndash, 800 &mu, g) should be loaded with GTP&gamma, S as a positive control for the pull-down assay. The following reaction details how to load endogenous RhoA with the nonhydrolysable GTP analog (GTP&gamma, S). 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 &ndash, 800 &mu, g of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer. b) Immediately add 1/100th volume of GTP&gamma, S (200 &mu, M final concentration). Under these conditions 5 - 10% of the RhoA protein will load with non-hydrolysable GTP&gamma, S and will be &ldquo, pulled down&rdquo, 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 GTP&gamma, S. Loading endogenous RhoA with GDP will inactivate RhoA and this complex will bind very poorly to Rhotekin-RBD beads.
Weight (grams)	11
Product Uses	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.
Material	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 RhC 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).
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 GTP?S (Cat. # BS01)
Figure 1 Legend	Figure 1. GST-Rhotekin-RBD protein purity determination. A 10 ug sample of PAK01 was
Figure 2 Legend	Figure 2: GST-Rhotekin-RBD protein purity determination. A 20 ug 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 ActivityThe 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 GTP?S (Cat. # BS01 )

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RT02-A.pdf

RT02-A-datasheet.pdf

RT02-A-safety-datasheet.pdf