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Solution Informationhelp
Enzyme: Dual specificity phosphatase Cdc25B
inhibitor: BDBM31757
substrate: n/a
 
Solution Type: Aqueous
pH at Preparation: n/a
Temp. Prep.: n/a
Comments: CDC25B Catalytic Domain Phosphatase Activity Protocol for 384-well Dose Response Assays. Description: Human Cdc25B Catalytic domain with His-tag, expressed in BL21(DE3) competent cells and purified using Talon Superflow Metal Affinity Resin. Recommended Usage: Screen at 3.126 ug/mL (15.63 ng/well). Storage: 10uL aliquots stored at -80 ◦C. Phosphatase Activity Assay Protocol Solutions: 10x Assay Buffer: 300mM TRIS pH 8 750 mM NaCl 10mM EDTA 1.5x Assay Buffer + BSA + DTT: Dilute 10X assay buffer to 1.5X assay buffer and add 0.099% BSA and 3mM DTT. OMFP (Sigma): Prepare a 50x stock of 0.002 mol/L by dissolving 1.05 mg OMFP in 1.0 mL DMSO. Immediately before the assay add 840 uL of 0.002 mol/L solution OMFP to 13.16 mL H2O, vortex, and immediately transfer 5 uL to all wells in assay plate using the Vprep. OMFP must be made fresh daily, and the assay concentration of OMFP will be near the Km of Cdc25B CD (~40 uM). Assay Stop Solution: 2mM Sodium Ortho-Vanadate (50
 
 

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Last update November 1, 2007
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