For more specific technical tips please view our product pages and datasheets.
The GTP loading method uses EDTA to remove MgCl2 and then GTP nucleotide is added to allow the exchange. Then MgCl2 is added back to lock in the GTP. Two datasheets give the experimental details (cat# RH01 and BK100). The RhoGAP assay biochem kit also provides useful information (cat# BK105).
If you are looking to measure the absolute GTP binding ability of a small G-protein, then first you must treat with EDTA, then perform size exclusion chromatography to remove all nucleotides. Then you can add back tritiated GTP and lock the nucleotide in with MgCl2, followed by additional size exclusion chromatography to remove any free tritiated GTP. Then, count the protein fractions in a scintillation counter. Some researchers have used DEAE discs instead of size exclusion chromatography to measure a small G-protein's GTP binding ability. Please search Methods In Enzymology and GTP binding for these method papers that were published between 1980-2000.
Answer 1: Cytoskeleton provides multiple options for measuring Rho protein in extracts from cells or tissue samples. Measuring Rho protein is typically discussed in terms of total and active (GTP-bound) levels of Rho.
To measure total Rho levels, we offer the RhoA ELISA (Cat. BK150) which has a linear range of 1 to 30 ng and a cv = 4%. An alternative is to use the anti-RhoA antibody to probe a blot of extracts, although this method is less accurate, it is useful for 1 to 5 samples.
To examine the levels of active Rho in extracts, Cytoskeleton offers the GLISA series of Activation assay kits. These kit contain all the reagents necessary to perform the assay from Lysis buffer through to protein assay and signal developing reagents. The kits are in 12 x 8-well strip format which allows you to use just 2 or 4 wells at a time. The results are highly reproducible and more accurate (cv = 13%, plus 8 fold linear range) than the conventional pulldown kits described next. Cytoskeleton also offers pulldown assays (e.g. BK036) which are packaged with all the necessary reagents, buffers and glutathione beads coupled to a Rho effector protein to complete the assay, in this format activated Rho levels are analyzed by western blot analysis which has cv's from 15 to 30% and a 3 fold linear range.
Conveniently, the GLISA and ELISA complement each other because the same lysate can be used with both kits. The G-LISA activation assays are often a better option than the traditional pull-downs as G-LISAs offer increased sensitivity, truly quantitative results, and the ability to process up to 96 samples at one time in less than 3 hours.
Active Rho levels in cells and tissues can be modulated by our G-switch line of reagents. Cytoskeleton offers indirect (Cat. # CN01) and direct (Cat. # CN03) Rho activators as well as a Rho inhibitor (Cat. # CT04). The direct activator and inhibitor are cell-permeable and robustly activate or inhibit RhoA activity, respectively, in a multitude of cell lines.
Answer 2: Cytoskeleton, Inc. offers a wide variety of kits and products that allow a thorough investigation of how a protein of interest affects small G-protein localization and function. We have highly-specific antibodies to RhoA (Cat. # ARH03), Rac1 (Cat. # ARC03) and Cdc42 (Cat. # ACD03) that can be used to examine changes in GTPase localization or expression levels. We also offer a Total RhoA ELISA kit to quantify total RhoA levels with the most sensitive and accurate assay currently available (Cat. # BK150). To measure small GTPase activation levels, we offer the G-LISA activation assays for RhoA (Cat. # BK124), Rac1 (Cat. # BK128), Rac1,2,3 (cat# BK125), Cdc42 (Cat. # BK127) and RalA (Cat. # BK129). Results with these 96-well format kits (presented as 12 x 8-well strips) are highly accurate and reproducible. Also the traditional pulldown assays are available for Ras (Cat. # BK008), Cdc42 (Cat. # BK034), Rac1 (Cat. # BK035), RhoA (Cat. # BK036) and RalA (Cat. # BK040).
To further examine how the protein of interest can affect GTPase activity, we also offer our G-switch line of small G-protein modulators. With these reagents, GTPase activity levels in cells and tissues can be activated or inhibited. Cytoskeleton offers indirect Rho (Cat. # CN01) and Rac/Cdc42 (Cat. # CN02) activators and direct Rho (Cat. # CN03) and Rho/Rac/Cdc42 (Cat. # CN04) activators as well as a Rho inhibitor (Cat. # CT04). The direct activators and inhibitor are cell-permeable and robustly activate or inhibit GTPase activity, respectively, in a multitude of cell lines.
To measure how your protein affects the signal transduction pathways involved in activating (GEFs) and deactivating (GAPs) GTPases, Cytoskeleton offers a RhoGEF Exchange Assay biochem kit (Cat. # BK100) and a RhoGAP Assay biochem kit (Cat. # BK105), respectively. The RhoGEF assay measures nucleotide exchange on GTPases using a fluorescent nucleotide analog while GAP activity is assessed by measuring the amount of inorganic phosphate produced as a result of hydrolysis of GTP to GDP by the GTPase.
If you are using microinjection as a tool to study your protein of interest in cells, Cytoskeleton offers purified, biologically-active and tagged GTPases (wild-type, dominant-negative and constitutively-active) that can be micro-injected into cells to study how your protein affects the GTPase.