| Assay Method Information | |
| | Determination of Biochemical BTK |
| Description: | 1. Reagents and Materials: BTK recombinant protein: Invitrogen, Cat #PV3363;Z′-LYTE kinase test kit-tyrosine 1 peptide: Invitrogen, Cat #PV3190;384-well low-flange black flat-bottomed polystyrene NBS microplate, no lid, no sterilization: Corning, Cat #3575;96-well polystyrene conical-bottomed MicroWell plate, sealed with a lid: Thermo Scientific Nunc , Cat #277143;Envision multi-mode plate reader: PerkinElmer; Mixmate shaker: Eppendorf;TS-2102 shaking incubator: TENSUC; 2. Methods: Z′-LYTE biochemical assay employs a fluorescence resonance energy transfer (FRET)-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. Both ends of the short peptide substrate are labeled with two fluorescent groups to form a FRET paired combination. In the primary reaction (the Kinase Reaction), the kinase transfers the γ-phosphate of ATP to a single serine or threonine residue on the short peptide substrate. In the secondary reaction (the development reaction), the non-phosphorylated short peptides were recognized and cleaved by a site-specific protease (the development reagent). |
| Affinity data for this assay | |
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