Measurement of the anisotropy of fluorescence polarization is a powerful tool to characterize the local environment of biomolecules. Anisotropy is a measurement of a molecule’s rotation from its excitation until it’s emission and can be influenced by many factors including molecular weight, binding state, viscosity, and alignment between excitation and emission dipoles or intrinsic anisotropy. The anisotropy parameter, r, is calculated according to the following where I is the parallel intensity and I is the perpendicular intensity:
Fluorescence polarization (FP) assays have long been used to measure change in effective mass of a fluorophore in bulk solutions. In a typical assay a fluorophore conjugated compound binds to a larger molecule thus changing its FP or anisotropy. The ImageXpress® Velos System enables object-based anisotropy measurements for bead and cell binding assays as well as more traditional bulk solution assays.
In this application note we present performance data on a beadbinding assay using a model system of biotinylated Alexa FluorTM 488 dye (b-AF488, Molecular Probes), steptavidin (SA) protein (Prozyme) and SA coated 10 µm polystyrene beads (Spherotech). Assays were performed in glass-bottom 384 well plates (Matrical) and scanned with a 488nm laser. b-AF488 emission was measured using photomultipliers (PMTs) with 510-540 nm BP filters (Omega). Two channels were measured: one collecting emission polarized parallel to the excitation laser (Para) and one collecting emission polarized perpendicular to the excitation laser (Perp). All data was analyzed using the BlueImage analysis program and ExcelTM (Microsoft).
Resuts - Bulk Solution
ImageXpress Velos System performance with bulk solutions was measured by comparing anisotropy of b-AF488 in phosphate buffered saline (PBS) to two mixtures of b-AF488 + SA in PBS. The samples are listed in the table below:
|Sample Name||b-AF488||Amount of SA|
|Dye||40 µl of 5 nM||none|
|Dye + 1x SA||40 µl of 5 nM||4 µl of 10 µg/m|
|Dye + 5x SA||40 µl of 5 nM||4 µl of 50 µg/ml|
Single well measurements were repeated 7 times over a 15 minute period. The anisotropy of b-AF488 in PBS was internally standardized to a value of 0.018. A plot of the average r over the 7 repeats for the first two solutions is shown in Figure 1. The error bars represent one standard deviation of the measurements. No change observed between the second and third solutions indicating saturation of binding. As expected, the anisotropy of the low molecular weight dye molecules increased upon binding to the higher molecular weight SA. Excellent differentiation between the two states was observed.
Results - Bead Binding
To show the effect of binding on the b-AF488 anisotropy, 20 µl of a 1:200 solution of SA coated beads were dispensed into a single well (see Figure 2). The sample was scanned to provide background signals. Some autofluorescence of the polystyrene beads was
observed. Subsequently, 20 µl of 10nM b-AF488 was added and series of 7 measurements were made over a 10 minute time course. The bead fluorescence intensity and anisotropy were measured by thresholding in BlueImage, extracting the bead specific data, and then calculating the average parameters over the bead population. Approximately 70 beads were measured in the field of view ofwell image and then calculating the parameters from the remaining pixels. Fluorescence intensity data for both the beads
and dye measured in Para and Perp channels is shown in Figure 3. The bead intensity steadily increases over a 6-8 minute time course and then saturates. This is consistent with the b-AF488 dye binding to the SA coated beads. A slight decrease in the Dye signal is shown due to the decrease in dye concentration after binding to the SA coated beads. A binding rate can be calculated from the data, if desired, and provide a measurement of initial dye concentration. The anisotropy was calculated at point and the average value shown is shown in Figure 4. The anisotropy of the bound dye was internally standardized to a value of 0.18 and the dye in solution to a value of 0.018. The error bars on the bead data points represent one standard deviation of the mean of the
bead population at each time point. The measured anisotropy of the bound dye was found to be very stable over the time course indicating that the microenvironment of the bound fluorophore did not change. Bead anisotropy in individual wells was measured over a full 384-well plate to asses the capability of the ImageXpress Velos System. Approximately 100 beads were analyzed per well and the average anisotropy value was calculated. The results of this analysis are shown in Figure 5. Excellent variation was observed with a full plate CV of 1.5
These results demonstrate that the ImageXpress Velos System can measure changes in bulk solution anisotropy as well as object specific anisotropy. Assays based on changes in anisotropy are enabled by this platform. In addition, anisotropy can be used as a gating parameter to enhance the robustness of more standard fluorescence intensity assays. Time course measurements for determination of kinetic rates are also enabled by the ImageXpress Velos System. Anisotropy measurements of cells are in development to enable assays for membrane binding and translocation as well as FRET based assays. The unique optics and scanning engine of this platform enables simple “plug and play” applications to meet the needs of both academic and biotechnology screening.