Apart from Activation Assays, Cytoskeleton provides some innovative kits for studying GEFs and GAPs. In particular the BK100kit is useful for measuring GTP or GDP exchange in Rho family members, and it can also be used to study a new GEF or a new GEF/small G-protein combination. The Rho GAP kit, Cat. # BK105, measures the release of phosphate from GTP hydrolysis by a Rho family protein which is accelerated by the GAP protein provided in the kit.
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Cytoskeleton's products have been cited hundreds of times over the past 18 years. Please contact Cytoskeleton's Technical Support at firstname.lastname@example.org for the latest citations.
RhoGAP Assay Biochem Kit (Cat. # BK105)
Zhang et al., 2010. A Novel Testis-specific GTPase Serves as a Link to Proteasome Biogenesis: Functional Characterization of RhoS/RSA-14-44 in Spermatogenesis. Mol. Biol. Cell. 4312–4324.
Rothenfluh et al., 2006. Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms. Cell. 127, 199–211.
RhoGEF Assay Biochem Kit (Cat. # BK100)
Moey et al., 2011. Ginseng (Panax quinquefolius) attenuates leptin-induced cardiac hypertrophy through inhibition of p115RhoGEF-RhoA/ROCKdependent MAPK pathway activation. J. Pharmacol. Exp. Ther. DOI:10.1124/jpet.111.182600.
Kim et al., 2009. Dipeptidyl Peptidase IV Inhibition With MK0431 Improves Islet Graft Survival in Diabetic NOD Mice Partially via T-Cell Modulation. Diabetes. 58, 641-651.
Li et al., 2009. PLC-gamma1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell Migration. Mol. Endocrinol. 23, 901-913.
Question 1: I'm expecting to see only a small difference in RhoA activation. What is the best way to measure this and what sensitivity can I expect?
Answer 1: To detect small differences in RhoA activation between control and treated conditions, we recommend either the absorbance or luminescence-based RhoA G-LISA activation assays (Cat. # BK124 or BK121, respectively). The absorbance-based RhoA G-LISA (Cat. # BK124) has a detection limit of 0.050 ng activated RhoA and a linear detection range of 0.05 – 2 ng activated RhoA. The coefficient of variation (8 replicates) is 12%. The luminescence-based RhoA G-LISA (Cat. # BK121) is recommended for use with 3-D cultures grown in matrigel or collagen matrices because of the additional sensitivity this kit offers. The luminescence-based RhoA G-LISA has a detection limit of 0.025 ng activated RhoA and a linear range of 0.025-1 ng activated RhoA.
Question 2: I have a purified protein that is possibly a Rho effector and a microtubule binding protein. How do I approach this project?
Answer 2: A good reference we recommend as a guide for studying such a protein is: Palazzo et al., 2001 (Nat. Cell Bio. mDia mediates Rho-regulated formation and orientation of stable microtubules. 3, 723-729). In general, domain cloning should be utilized and then the RhoA binding activity of each domain would be measured with the positive control RhoA protein (enough for 96-assays) included in each RhoA GLISA kit (Cat. # BK124). The presence of effector domain should reduce the RhoA signal because it will compete for RhoA protein in the well. To examine binding between the purified protein and microtubules, we recommend using the microtubule binding protein spin-down assay biochem kit (Cat. # BK029).
For more information, click on the Documents tab above and see the datasheet, or contact Technical Support at email@example.com.