| Solution Information | help | |
| Enzyme: | Serine/threonine-protein kinase PLK1 | |
| inhibitor: | BDBM32006 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | PLK1-PBD Binding Assay protocol Reagents & Materials: The His6-GST-PBD protein, FITC-labeled phospho-peptide and unlabeled phospho-peptide were provided to the PMLSC by the assay provider (Dr. Michael Yaffe, MIT). 10X Assay Buffer: 500 mM Tris, 2 M NaCl, 20 mM DTT , 0.05 % Tween 20, pH 8.0. Tris HCl 60.57g NaCl 116.88g Tween 20 500uL(0.05%) dH2O 800mL pH to 8.0 with NaOH add dH2O to 1000mL DTT: 200 mM Stock in -20 ◦C. His6-GST-PBD protein: 150 ng in 5 uL of 3X assay buffer. 50 uL aliquot of His-GST-PBD @ 70 mg/mL provided by the assay provider was diluted to 10 mg/mL by adding 300 uL of 1X assay buffer; 50 mM Tris, 200 mM NaCl, 2.0 mM DTT , 0.005 % Tween 20, pH 8.0. The protein was then aliquoted and stored at -80C. To prepare 150 ng His-GST-PBD in 5 uL of 3X assay buffer add: 160 uL of 10 mg/mL His-GST-PBD + 16.0 mL of 10X assay buffer, + 1.6 mL of 200 mM DTT + 35 mL of H2O. FITC-Phospho-peptide : 30 uM stock of FITC-Phospho-peptide in 10 mM Tris pH 8.0 + 1mg/mL BSA. Add | |
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