| Solution Information | help | |
| Enzyme: | Protein RecA | |
| inhibitor: | BDBM50303409 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | GFP refolding assay in Dose (counter screen) 1) Dispense positive control Dispense 100 nL of positive control ZnCl2(60mM) using Combi nl in respective wells according to plate design to 1536-well assay ready plates (Aurora 00027830) that contain 22.5 nL/well of 10 mM compound in 8 doses with 3fold dilution(generated by Echo acoustic dispenser). 2) Dispense denatured GFP Add 3 uL/well of denatured GFP(0.6uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes. 3) Dispense TCEP to initiate the reaction Add 3ulL/well of TCEP(2mM) with Combi nL (Thermo), incubate at room temperature for ~18 hours 4)Centrifuge Centrifuge the plates at 1000 rpm x1min 5) Read on Envision for Fluorescence(Ex.405nm/Em.510nm) Solutions: Intein buffer 20mM NaPi 7.0 500mM NaCl 500mM L-Arginine.HCl 0.01% Triton-100 1% DMSO ZnCl2 (in H2O, Sigma 229997-50G) 60mM ZnCl2 TCEP (in intein buffer, Sigma 646547) 2mM TCEP denatured GFP- (in intein buffer, provided by Henry Paulus's lab) 0 | |
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