Measurement by Spark 20M / Spark 10M Multimode microplate reader

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1 Application Note Fluorospark Kinase/ADP Multi-Assay Kit Measurement by Spark 20M / Spark 10M Multimode microplate reader Introduction Fluorospark Kinase/ADP Multi-Assay Kit Phosphorylation and dephosphorylation of proteins in signal transduction play a critical role in physiological regulation as immune response, oncogenesis, differentiation, apoptosis and cell proliferation. Kinase, which is the enzyme catalyze the phosphorylation of proteins, lipids and other molecules, is closely related to the variety of the diseases including a cancer and to be a main drug discovery target today. Fluorospark Kinase/ADP Multi-Assay Kit is a simple and highly sensitive ADP quantitation kit through by enzymatic coupling reaction. The generated ADP through kinase reaction has been quantified as red fluorescent resorufin. By the quantification of resorufin, this kit can easily quantify the kinase activity with high sensitivity. Furthermore the accuracy of the measured value is higher than previous bioluminescent methods. This kit can utilize for not only the endpoint assay but also the real-time assay. This application note explains our confirmation of the measuring capability. ADP standard curves were generated using the Fluorospark Kinase/ADP Multi-Assay Kit by the Spark 20M Multimode microplate reader, and a comparison study on sensitivity, accuracy, and measuring time was conducted. As a result, fast and highly accurate measuring work was confirmed. 1

2 Protocol of Fluorospark Kinase/ADP Multi-Assay Kit Figure 1: Principle of Kinase activity measurement by Fluorospark Kinase/ADP Multi-Assay Kit Materials and Method Instruments: Spark 20M Multimode microplate reader / Spark 10M Multimode microplate reader (Tecan) Reagents: Fluorospark Kinase/ADP Multi-Assay Kit (Wako Catalog No ) Microplate: 384 well microplate, flat-bottom, black, small volume (Greiner Catalog No ) Assay Procedure First, an ADP dilution series was prepared at ATP-to-ADP Conversion Rate 0-100% ([ATP]+[ADP]=10μM). Then, a mixture solution (5 μl/well) was applied to each well (Table 1). Next, 2x Detection Solution was prepared at the time of use by mixing Substrate Solution, Enzyme Solution, Resazurin Solution and Reductant and Reductant Blocker, which are included in Fluorospark 2

3 Kinase/ADP Multi-Assay Kit. 5 μl each of the solution was added to each well that contains the solution of ADP dilution series. Then, incubation was implemented for 30 minutes at room temperature. Next, the fluorescence intensity was measured by using a Spark 20M or Spark 10M Multimode microplate reader, and ADP standard curves were generated. In addition, the Z -factor was calculated at ATP-to-ADP Conversion Rate 10% ([ATP]+[ADP]=10μM) to confirm the compatibility of the assay series with HTS. The following formula was used to calculate the Z -factor. Z -factor = 1 - (3 SDH + 3 SDL)/(AVEH AVEL) SDH and SDL: Each standard deviation at ATP-to-ADP Conversion Rate 10% and 0% AVEH and AVEL: Each average value at ATP-to-ADP Conversion Rate 10% and 0% The following shows the parameter and equipment setting for Z -factor measuring. Measuring Terms Setting for Spark 20M and Spark 10M Result Figure 2 shows the ADP standard curves by Spark 20M and Spark 10M. On either Spark 20M or Spark 10M, linearity of ATP-to-ADP Conversion Rate 0-100% was confirmed for both the filter method and monochrometor method. Also, adequate linearity was found even at low concentration range (ATP-to-ADP Conversion Rate 0-10%). This shows that accurate quantitation can be made even with low ADP production. 3

4 In addition, the Z -factor and measuring time were compared with various numbers of flashes (5, 10, 20, and 30 times) (Table 2). As the result, all Z -factor were 0.5 or above according to either number of flashes. This shows that stable and highly accurate results are attainable even with a low number of flashes. With Spark 20M, it was possible to attain a 0.8 level for the Z -factor even with 5 flashes. Furthermore, the measuring time using Spark 20M was shorter than using Spark 10M. It is considered that Spark 20M is the optimal measuring device for HTS. Table 1: ADP dilution series <Conversion rate: 0-100%> <Conversion rate: 0-10%> ADP-Low concentration range 4 Figure 2 : Standard curves of ADP with Fluorospark Kinase/ADP Multi Assay Kit

5 Comparison of Z -factor (n=96) The nearer 1 the Z -factor is, the more sensitive and lower variable assay system this kit is. Comparison of measurement time (sec.) Table 2 : Z -factor and the measurement time at Spark 20M and Spark 10M Conclusion Spark 20M and Spark 10M are measuring systems that boast superior assay accuracy. This study shows that highly sensitive and highly accurate ADP measurements can be accomplished by using these systems with the Fluorospark Kinase/ADP Multi-Assay Kit. In particular, Spark 20M is the optimal measuring device for HTS, which requires fast and highly accurate measuring work for a large number of plates. Reference: Evelopment of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescene detection, Kumagai, K., Kojima, H., Okabe, T., Nagano, T.: Anal. Biochem., 447, (2014). LINK: Wako Pure Chemical Ind., Ltd , Doshomachi 3-Chome Chuo-Ku, Osaka , Japan Tel: Fax: Online Catalog: Wako Chemicals USA, Inc Bellwood Road Richmond, VA 23237, USA Toll-Free (U.S. only): Tel: Fax: Wako Chemicals GmbH Europe Office Fuggerstraße 12, D Neuss, Germany Tel: Fax: