| Assay Method Information | |
| | CSF1R Enzymatic Inhibitory Assay |
| Description: | The compounds were supplied in a 10 mM DMSO solution, and enzymatic CSF1R inhibition potency was determined by Invitrogen (TermoFisher) using their Z′-LYTE® assay technology (B. A. Pollok, B. D. Hamman, S. M. Rodems, L. R. Makings, Optical probes and assays, WO 2000066766 A1, 5-5-2000.). The assay is based on fluorescence resonance energy transfer (FRET). In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Thus, phosphorylation of FRET-peptides suppresses cleavage by the development reagent. Cleavage disrupts FRET between the donor (i.e.,coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A ratiometric method, which calculates the ratio (the emission ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate inhibition. All compounds were first tested for their inhibitory activity at 500 nM in duplicates. The potency observed at 500 nM was used to set starting point of the IC50 titration curve, in which three levels were used 1000 or 10000 nM. The IC50 values reported are based on the average of at least 2 titration curves (minimum 20 data points), and were calculated from activity data with a four parameter logistic model using SigmaPlot (Windows Version 12.0 from Systat Software, Inc.) Unless stated otherwise the ATP concentration used was equal to Km (ca 10 mM). The average standard deviation for single point measurements were <4%. |
| Affinity data for this assay | |
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