Compatible Readers
Randy Hoffman1, Megan Buros1, Kevin Kupcho1 and E.J. Dell2
1Invitrogen Corporation, Madison, WI, USA; 2BMG LABTECH, Durham, NC, USA
Introduction
Protein Kinase C (PKC) enzymes are a diverse family of enzymes that under specific signaling conditions phosphorylate proteins on serine and threonine amino acids. PKCs are involved in many cellular functions including neuronal activity, cell growth, cell proliferation, and cell movement, as well as in several diseases including cardiovascular, cancer, diabetes, and Alzheimer’s (1). Currently, ten different human isoforms of PKC have been cloned; however, their tissue specific roles have yet to be determined. Furthermore, both the number of receptors that activate PKCs and the number of PKC specific substrates continue to grow (2). Therefore a TR-FRET screening assay, such as the one described here, can aid in further elucidating PKC’s function.
The LanthaScreen™ TR-FRET platform from Invitrogen uses terbium as the donor group and fluorescein as the acceptor species. The terbium/fluorescein-based LanthaScreen™ configuration has several advances; these include simpler assay optimization, faster kinetics of complex formation, avoidance of steric problems, as well as the cost and lot-to-lot consistency of directly labeled substrates.
BMG LABTECH’s PHERAstar is a multifunctional plate reader that combines rapid plate reading necessary for high throughput screening (HTS) with the enhanced performance and sensitivity needed to read small fluid volumes. The PHERAstar has been designed to read all HTS detection modes (fluorescence intensity, time-resolved fluorescence, fluorescence polarization, luminescence, and absorption) in all plate formats up to 1536 wells. The PHERAstar uses a unique application-specific module in conjunction with an optical reading head featuring five photomultiplier tubes that can simultaneous measure two emission signals at any desired wavelength. This optical design provides for outstanding sensitivity and accuracy in fluorescence and luminescence assays, and the simultaneous measurement minimizes the read time for assays.
Assay Principle
A fluorescein labeled PKC substrate peptide is incubated with PKC and ATP. Subsequently, a terbium labeled antibody that binds to the phosphorylated form of the substrate is added. When the antibody interacts with the phosphorylated substrate, FRET will occur between the terbium label (emits at 490 nm) and the fluorescein moiety (emits at 520 nm) (Figure 1). Measurements are taken at 490 and 520 nm and the 520/490 ratio is plotted versus the concentration of enzyme to determine an EC50 for the enzyme and its substrate.
Fig. 1: LanthaScreen™ TR-FRET Assay Principle
Materials and Methods
Fig. 2: PHERAstar Multimode Microplate Reader
PKC Titration with LanthaScreen™ Technology
Multiple PKC isoforms were titrated to determine optimal kinase concentrations for screening. The following protocol is an example of the conditions used to determine the EC50 of PKCα and its substrate. A dilution series of PKC, starting at a final concentration of 2.0 µg/ ml, was incubated in the presence of 250 nM fluorescein-labeled PKC substrate and 20 µM ATP in a total volume of 10 µl in a black Corning® low volume 384-well plate (Corning #3676). After a 90- minute incubation at room temperature, 10 µl of TR-FRET dilution buffer containing 2X EDTA (20 mM) and 2X Tb-PKC antibody (1.0 nM) was added and mixed to create a final volume of 20 µl per well, a final 1X EDTA concentration of 10 mM, and a final 1X antibody concentration of 0.5 nM. After incubating for 60 minutes at room temperature, the plate was read on the BMG LABTECH PHERAstar. Each data point represents the average of three wells.
Data Analysis
Signal-to-noise ratio is calculated using the formula:
S:N = (|µp - µn|) / (sp² + sn²) ½
Z’-factor, a standard for evaluating HTS methods, is calculated using the formula:
Z’ = 1 – [(3sp + 3sn) / (|µp - µn|)
where µp = mean of “positive control” (max ratio), µn = mean of “negative control” (min ratio), and sp and sn = the corresponding standard deviations.
Results and Discussion
Figure 3 shows representative kinase titration curves for PKCα and PKCÇ.
Fig. 3: LanthaScreen™™ on the PHERAstar: PKCα and PKCÇ.
Table 1 shows statistical data for all ten kinases tested.
Table 1: LanthaScreen™ on the PHERAstar: statistics for PKC isofrom titrations.
All ten PKC isoenzymes were titrated in a similar manner as PKCα. Regardless of “fold difference” or signal-to-noise ratios, all assays provided Z’ values far greater than 0.5. This is due to the use of FRET-based, ratiometric data analysis, which leads to low standard deviations of the replicates. Both hill slope and R2 measurements fall within acceptable limits. R2 values approaching 1.0 indicate a perfect curve fit.
The results presented here demonstrate that LanthaScreen™ PKC assays are sensitive in that they require sub ng/ml kinase concentrations, thereby keeping overall screening costs down. Furthermore, the assays are robust with Z’ values greater than 0.7, which provides confidence that the assay data is meaningful.
Conclusion
Invitrogen’s terbium-based LanthaScreen™ has several unique properties:
1. The ratiometric nature of LanthaScreen™ eliminates well to well variation.
2. The time-resolved nature of LanthaScreen™ assays allows for the use of fluorescein without the associated drawbacks of compound interference.
3. The ability to use fluorescein simplifies assay development and costs.
BMG LABTECH’s PHERAstar microplate reader provides the ideal platform to simplify Invitrogen’s LanthaScreen™ assay development. With its dual wavelength emission detection and five photomultiplier tubes (PMTs), the PHERAstar provides the speed and sensitivity needed to take full advantage of Invitrogen’s LanthaScreen™ technology. Furthermore, BMG LABTECH has optimized its TR-FRET PMTs to allow for the most sensitive LanthaScreen™ detection system on the market; and BMG has designed an optic module (Figure 4) specifically for Invitrogen’s LanthaScreen™, thereby making assay setup simple.
Fig. 4: LanthaScreen™optical module with dual wavelength detection
References
1. Battaini F, Pascale A. Protein kinase C signal transduction regulation in physiological and pathological aging. Ann NY Acad Sci. 2005 Dec;1057:177-92. Review.
2. Way KJ, Chou E, King GL. Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci. 2000 May;21(5):181-7. Review.
BMG LABTECH GmbH, Hanns-Martin-Schleyer-Str. 10, D-77656 Offenburg/Germany
Phone: +49 781 96968-0, Fax +49 781 96968-67, Impressum
Copyright ©1995-2008
All Rights Reserved.
