Overcoming the hurdle of fluorescent compounds in kinase screening: a case study.
In the field of screening in general and especially in the kinase area, taking into consideration throughput and cost, fluorescence- and luminescence-based assays have been developed as alternatives to radioactivity-based assays. However, fluorescence-based technologies are not devoid of pitfalls. One of the main problems is interference from autofluorescent compounds and the incidence of false-positives as exemplified here with a fluorescence polarization (FP)-based assay. Using the scintillation proximity assay as the in-house standard, we assessed several alternatives to radioactive methods, namely, the amplified luminescent proximity homogeneous assay screen (ALPHAScreen, Perkin-Elmer Life Sciences, Boston, MA), enzyme fragment complementation, FP, and nanofluidics-based fluorescence intensity. Data comparing the sensitivity, robustness, relative sensitivity to autofluorescent compounds, enzyme consumption, and relative costs of each assay for one common kinase are presented. Results obtained seem to favor the nanofluidics mobility shift assay as a method of choice, followed by the direct FP approach, using generic high-molecular-weight phosphate group-binders.
Perrin
Dominique
D
Molecular Screening and Cellular Pharmacology Department, Serono Pharmaceutical Research Institute, Geneva, Switzerland. dominique.perrin@serono.com
Martin
Thierry
T
Cambet
Yves
Y
Frémaux
Christèle
C
Scheer
Alexander
A
D016231:Fluorescence Polarization Immunoassay; D005456:Fluorescent Dyes; D008163:Luminescent Measurements / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D017346:Protein Serine-Threonine Kinases