| Assay Method Information | |
| | LanthaScreen Eu Kinase Binding Assay |
| Description: | The reagent was prepared through a series of dilutions of the tracer. The tracer was first diluted to 3000 nM by adding 3.6 μL of 50 μL stock tracer to 56 μL of 1× Kinase Buffer A. 50 μL of 1× Kinase Buffer A was added to 5 wells in each of two columns of a 96-well plate. 50 μL of the 3000 nM tracer was added to well A1 and mixed. 50 μL of solution was removed from A1 and transferred to A2 and mixed. 50 μL of the solution in well A2 was removed and transferred to well B1 and mixed. This protocol was repeated nine times to the desired concentration. The kinase/antibody solution was prepared at 15 nM kinase, 6 nM antibody, and 6 nM Eu-Streptavidin. Both the antibody tube and Eu-Streptavidin tube were centrifuged at approximately 10,000×g for ten minutes, and the desired volume was aspirated from the top. The volume of reagents added to Kinase Buffer A were calculated using the equations provided in the LanthaScreen® Eu Kinase Binding Assay Validation Packet. 30 μM staurosporine (“competitor solution”) was prepared by diluting 30 μL of 1 mM staurosporine (from a stock in DMSO) into 970 μL Kinase Buffer A. A 3% DMSO control solution was prepared by adding 30 μL DMSO to 970 μL Kinase Buffer A.5 μL of each concentration of serially diluted tracer was added to six replicate assay wells in a 384-well plate. 5 μL of competitor solution was added to three wells for each tracer concentration. 5 μL of DMSO control solution was added to the other three wells for each tracer concentration. 5 μL of kinase/antibody solution was added to all wells, and the plate was incubated at room temperature for 60 mins. |
| Affinity data for this assay | |
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