Compatible Readers
Randy Hoffman1, Megan Buros1, Kevin Kupcho1 and E.J. Dell2
1Invitrogen Corporation, Madison, WI, USA; 2BMG LABTECH, Durham, NC, USA
Introduction
Tyrosine kinases (TKs) are a diverse family of enzymes that under specific signaling conditions phosphorylate proteins on the amino acid tyrosine. TKs are involved in many cellular functions such as cell division and cell proliferation, as well as in several diseases including cancer and diabetes. TKs can be divided into two subcategories: 1) receptor-linked tyrosine kinases, which include epidermal growth and insulin receptors; and 2) cytoplasmic/nonreceptor tyrosine kinases, which include Src and Fyn(1). In humans, 32 non-receptor TKs and 58 receptor TKs have been currently cloned. Tyrosine kinases play important roles in cell growth, thereby making them important drug targets in cancer therapy(2). For that reason a TR-FRET screening assay, such as the one described here, can aid in elucidating specific inhibitors for tyrosine kinases.
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 Design
A fluorescein labeled poly-GT or poly-GAT (Glu, Ala, Tyr) substrate is incubated with a tyrosine kinase 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
Tyrosine Kinase Titration with LanthaScreen™ Technology
To demonstrate the functionality of a universal tyrosine kinase assay using LanthaScreen™, both fluorescein-poly-GAT (Glu, Ala, Tyr) and fluorescein-poly-GT (Glu, Tyr) substrates were used with terbium labeled PY20 antibody. Figure 3 shows titration curves using both substrates for two tyrosine kinases, IGF-1R and ZAP-70. Table 1 shows the EC50 calculations derived from 24 different kinase titrations using both substrates. A dilution series of kinase was incubated with 400 nM fluorescein labeled substrate and 200 µM ATP in a total volume of 10 µl in a black Corning® low-volume 384-well plate (Corning #3676). After 60 mins of incubation at room temperature, 5 µl of TR-FRET dilution buffer containing 4X EDTA (60 mM) was added. Next, 5 µl of TR-FRET dilution buffer with 4X antibody (8 nM) was added and mixed to create a final volume of 20 µl per well, a final 1X EDTA concentration of 15 mM, and a final 1X antibody concentration of 2 nM. After 60 mins of incubation at room temperature, the plate was read on the BMG LABTECH PHERAstar. Each data point represents the average of four wells.
Results and Discussion
Figure 3 shows kinase titration curves for ZAP-70 (a, b) and IGF-1R (c,d) using both poly-GAT (a, c) and poly-GT (b, d). Table 1 shows statistical data for all 24 tyrosine kinases tested.
Fig 3: LanthaScreen™ on the PHERAstar: ZAP-70 (a, b) and IGF-1R (c, d) titrations.
Table 1: EC50 calculations from tyrosine kinase titrations using both poly-GAT and poly-GT substrates
The results show that use of a fluorescein-labeled substrate common for many TKs (poly-GT or poly-GAT) allows for the development of a “universal” TK assay using the LanthaScreen™ format. For many of the TKs shown, EC50 values are similar for both substrates. But for several kinases, especially the FGFRs, EC50 values are much lower for the poly-GT substrate. This may indicate that the poly-GT substrate provides a more sensitive assay compared to the poly-GAT. However, on average, the poly-GAT substrate provided an increased dynamic range compared to poly-GT (~50 fold compared to ~17 fold). Therefore, depending on the kinase, the customer can choose which is more important: using less kinase or having increased dynamic range.
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. Hubbard SM and Till, JH. Protein Tyrosine Structure and Function.
Ann Rev of Biochem. 2000 July; 69:373-398.
2. Arora A and Scholar EM. Role of tyrosine kinase inhibitors in cancer
therapy. J Pharmacol Exp Ther. 2005 Dec;315(3):971-9.
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