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Question 1: What is the best way to measure GEF activity from a cell lysate?
Answer 1: First, the GEF protein must be immunoprecipitated from the cell lysate with either an antibody to the protein or to a tag (His, GST, etc.) that has been conjugated to the protein of interest. After eluting your protein of interest from the beads, the protein&rsquo, s GEF activity can be measured using Cytoskeleton&rsquo, s RhoGEF Exchange Assay Biochem Kit ((Cat. #BK100). Cytoskeleton Inc. has developed a mant fluorophore-based GEF assay designed for characterizing GEFs and identifying GEF inhibitors. This kit contains human Cdc42 (Cat. #CD01), Rac1 (Cat. #RC01) and RhoA (Cat. #RH01) proteins and the GEF domain of Dbs (Cat. #GE01) as a positive control GEF for Cdc42 and RhoA. The kit also comes with a 384-well and 96-well plate along with exchange buffer that contains the mant-GTP. Once bound to GTPases, the fluorophore emission intensity of mant-GTP increases approximately 1.1 to 2.0 fold depending on the protein. Therefore, the enhancement of fluorescent intensity in the presence of small GTPases and GEFs will reflect the respective GEF activities of known or unknown proteins. We recommend titrating the concentration of your GEF protein to optimize its activity. We suggest a titration range of 0.01-1 µ, M. A citation that used our GEF assay kit with an immunoprecipitated protein is Kakiashvili et al., 2009. GEF-H1 mediates tumor necrosis factor-alpha-induced Rho activation and myosin phosphorylation: role in the regulation of tubular paracellular permeability. J. Biol. Chem. 284, 11454-11466.
Question 2: I want to use the RhoGEF Exchange Assay kit with a 384 well plate. Is this possible?
Answer 2: Yes, Cytoskeleton&rsquo, s mant fluorophore based GEF assay ((Cat. #BK100) is suitable for a 384 well high-throughput screen format. Up to 130 reactions per GTPase can be achieved if a 384-well low volume black round bottom plate (Corning Cat# 3676) is used. This type of 384 well plate is strongly recommended due to the greater signal/noise ratio. Do not use clear plates since that will give you significant background noise. We provide a 384 well plate and a protocol for this HTS format in the manual that comes with the kit.
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Alle Produkte sind nur für Forschungszwecke bestimmt. Nicht für den menschlichen, tierärztlichen oder therapeutischen Gebrauch.
BK100.pdf
BK100-datasheet.pdf
BK100-safety-datasheet.pdf
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