| Solution Information | help | |
| Enzyme: | Importin subunit alpha-1 | |
| inhibitor: | BDBM74735 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | 2041-02 RanGTP inhibitor-Counterscreen-FRET assay protocol The plasmid expressing YIC (a protein contains the importin-beta binding domain of importin-alpha flanked by CFP and YFP) is from assay provider#s lab and the protein is purified from E.coli by Macrolab at University of California at Berkley. The protein is purified from Ni chromatography due to His tag followed by gel filtration. The purity of the protein is assigned by SDS-PAGE, the function of the protein is assigned by its ability to bind to his-tagged importin-beta (Sigma, Cat R 3152) 1. The counterscreen in dose with YIC protein uses Assay Ready Plate (ARP). ARP contains 200nL/well of compound in 384-well plate (Corning, Cat 3573). The concentration series is started at 10mM with a dilution factor of 2 for 8 points 2. Dispense 40uL/well of Poscon solution to Poscon wells by Combi (Thermo Scientific). Poscon solution contains 62.5nM CFP-Ran, 20nM RCC1, 200uM GDP and 62.5nM YFP-Importin-beta in 1x Assay Buffer. 3. Dis | |
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