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Solution Informationhelp
Enzyme: Regulator of G-protein signaling 8
inhibitor: BDBM34524
substrate: n/a
 
Solution Type: Aqueous
pH at Preparation: n/a
Temp. Prep.: n/a
Comments: Protocol used for the dose response assay is similar to the one used for screening, with the exception that compounds were tested at 9 different concentrations. Each component of the multiplex assay consists of a streptavidin functionalized polystyrene bead, a biotinylated RGS-fusion protein target (bio-RGS, five total, supplied by project collaborator), and a fluorescent probe, AlexaFluor488-labeled Galphao protein. Six bead sets are used, including one unlabeled bead set and five sets that are labeled to different intensities with red fluorescence that are fluorescent in APC-Cy7 (FL9, 750 nanom LP) channel at 635 nanom excitation (Spherotech product numbers SVPAK-5067-5B). Individual bead sets are coupled with a single bio-RGS protein by mixing beads and bio-RGS in bead coupling buffer (BCB; PBS, pH8.0 supplemented with 0.1% BSA). The mixture is then incubated overnight at 4oC under mild vortexing. The 5 bead sets (each with a bound protein) and an uncoated bead set (Scavenger bead
 
 

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Last update November 1, 2007
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