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Encompassing Amino Acids: Full Length
Application: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: CDK3/CyclinE2 activity was measured by using Histone H1 Substrate, diluted in distilled water to a working concentration of 1 mg/ml, in a [33P]-ATP based assay. Reaction was initiated by mixing increasing amounts of CDK3/CyclinE2 with 1250 pmoles of [33P]-ATP in 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 50 ng/µl BSA, 50 µM DTT and substrate at a final concentration of 200 µg/ml.The reaction was initiated by addition of 5 µl of [33P]-ATP Assay Cocktail (50 µM of [33P]-ATP with 50 µM ATP) to 20 µl of Reaction Mix (10 µl of diluted CDK3/CyclinE2) + 5 µl of Substrate Solution + 5 µl of distilled water), followed by a 15-minute incubation at 30°C. The reaction was terminated by spotting the reaction mixture on phosphocellulose P81 paper, air-dry and three 10-minute washes with 1% phosphoric acid solution. Radioactivity was measured in a scintillation counter. The corrected activity (RLU) was calculated by removing the blank value for each sample. The Kinase Specific Activity was calculated as follows: RLU / [(specific activity of [33P]-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in µg or mg)] * [(Reaction Volume) / (Spot Volume)]. The blank was determined from a "no substrate" sample by replacing the substrate solution with an equal volume of distilled water.
Description: Complex of recombinant human full length CDK3 (cyclin dependent kinase 3) and CyclinE2. Both constructs contain an N-terminal GST-tag. The complex was affinity purified and is active.
Format: Aqueous buffer solution
Formulation: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, and 25% glycerol
Genbank: CDK3: NM_001258
Cyclin E2: NM_057749
Purity: ≥90%
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal GST-tags
Uniprot: CDK3: Q00526
Cyclin E2: O96020
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
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