Off chip mobility shift assay

Kinase activity was measured by Off-chip Mobility Shift Assay in Carna Biosciences. The 4× substrate/ATP/metal solution was prepared with kit buffer (20 mM HEPES, 0.01% Triton X-100, 5 mM DTT, pH 7.5), and 2× kinase solution was prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 1 mM DTT, pH 7.5). The 5 μl of 4× compound solution, 5 ml of 4× substrate/ATP/metal solution, and 10 ml of 2× kinase solution were mixed and incubated in a well of a polypropylene 384 well microplate for 1 h at RT. Then, 70 ml of termination buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added to the well. The reaction mixture was applied to LabChip™ system (Perkin Elmer), and the product and substrate peptide peaks were separated and quantitated. The kinase reaction was evaluated by the product ratio calculated from peak heights of product (P) and substrate (S) peptides (P/(P+S)).

Kinase activity was measured by Off-chip Mobility Shift Assay(MSA) in Carna Biosciences. The enzyme was incubated with fluorecence-labeled substrate and Mg(orMn)/ATP. The phosphorylated and unphosphorylated substrates were separated and detected by MSA device. Specific operations were carried out according to the product manual. Where A equals negative control, B equals positive control and C equals test sample. Calculate the percent inhibition of compound as follows: Inhibition (%) = (1-(C-A)/(B-A)) x 100.