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| Solution Information | help | |
| Enzyme: | Dual specificity protein phosphatase 1 | |
| inhibitor: | BDBM31044 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials: 1.10X Assay Buffer: 300 mM Tris, 750 mM NaCl, 10 mM EDTA, pH 7.0. Trizma 47.28 g + NaCl 43.83 g +EDTA 3.722 g + dH2O 800mL, pH to 7.0 with NaOH, add dH2O to 1000mL. 2.DTT: 200 mM Stock in -20 ◦C. 3.BSA: 6.6% BSA Stock in -20 ◦C. 4.OMFP Substrate: 5.0 uL/well ~ 40 uM final (1% DMSO). Add 1.05 mg OMFP per 0.5 mL of DMSO and sonicate in water bath ~ 5 min to make a 4.0 mM stock of OMFP (100% DMSO). Immediately prior to the assay dilute the 4.0 mM OMFP to 120 uM in dH2O and transfer 5 uL to each well of a 384-well of the assay plate ~ 40 uM OMFP final (1% DMSO). 5.MKP-1 Enzyme: 5.0 uL/well ~ 250 ng/well. Partially purified MKP-1 enzyme at 685 ug/mL in 0.5 mL aliquots stored at -80 ◦C. 6.Compounds: 5.0 uL/well ~ 10 to 100 uM final (1% DMSO). Dilute compounds in dH2O to the appropriate concentration (30 to 300 uM, 3% DMSO) and transfer 5 uL per well. 7.Maximum Control: 5.0 uL/well of 3% DMSO in dH2O 8.Minimum Control: 5.0 uL/well of 300 mM NaOH in 3% DMSO in d | |
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