Assay Method Information

Assay Name:  In Vitro Kinase Screening Assay
Description:  A reaction buffer containing 200 mM Tris pH 7.5, 100 mM MgCl2 and 0.1 mg/ml BSA was added to 43 μM SRSF1Arg-Ser (RS) peptide (NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH) and 0.1 μg of purified SRPK1 (ProQinase, Freiburg, Germany). Candidate compounds were serially diluted from 10 μM to 0.001 nM and added to the reaction mixture, wells with omitted SRPK1 kinase and omitted compounds were also added as controls. All wells contained 1% DMSO (Fisher Scientific, Loughborough, UK). 1 μM ATP was added, wells minus ATP were used as background controls. The plate was then incubated at 30 °C for 10 minutes. An equal volume of Kinase-Glo (25 μL; Promega) was added to each well and the plate read for luminescence using a Fluostar Optima (BMG Labtech). Radioactive kinase assays were carried out by the MRC Dundee Kinase Centre. Lack of interference with binding to the SRPK1 substrate was carried out using a dose response curve from 0.5-30 μM.
Affinity data for this assay
 

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