Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

2015 ◽  
Author(s):  
Yang Pu ◽  
Robert R. Alfano
Keyword(s):  
Fluorescence Polarization ◽  
Near Infrared ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Polarization Spectroscopy

scholarly journals Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

2013 ◽  
Vol 85 (3) ◽  
pp. 589-608 ◽  
Author(s):  
Marcel Ameloot ◽  
Martin vandeVen ◽  
A. Ulises Acuña ◽  
Bernard Valeur
Keyword(s):  
Steady State ◽  
Fluorescence Anisotropy ◽  
Fluorescence Polarization ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Steady State Fluorescence ◽  
Technical Report ◽  
Accuracy Of Measurements ◽  
Solution Methods ◽  
Systematic Effects

After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.

scholarly journals Utilization of Fluorescence Polarization and Time Resolved Fluorescence Resonance Energy Transfer Assay Formats for SAR Studies: Src Kinase as a Model System

2004 ◽  
Vol 9 (6) ◽  
pp. 525-532 ◽  
Author(s):  
Miki Newman ◽  
Serene Josiah
Keyword(s):  
Energy Transfer ◽  
Small Molecule ◽  
Fluorescence Polarization ◽  
Src Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Sar Studies ◽  
Lead Identification

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA®), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (μM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIA® assays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of Ki = 0.01nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.

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