Kinase Assay Screening Kit
Fluorospark™ Kinase/ADP Multi-Assay Kit

Fluorospark^™ Kinase/ADP Multi-Assay Kit, which is jointly-developed with Drug Discovery Initiative, The University of Tokyo, is a simple and highly sensitive ADP quantitation kit for high throughput screening (HTS) with lower cost. This kit is also applicable to ATPase, Acetyl-CoA carboxylase kinase assays as well as kinase assay which generate ADP.

• 1. Features
• 2. Comparison with conventional (luminescent) method
• 3. Summary
• 4. Kit Contents

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< Endpoint assay >

• 5. Protocol of Endpoint assay
• 6. Performance
  
  
  • 6-1. Preparation of Standard Curve of ADP
  • 6-2. ADP Standard curve at the low substrate conversion rate
  • 6-3. Fluorescent Signal Stability
• 7. Data
  
  
  • 7-1. Calculation of Z'-factor
  • 7-2. Inhibition Curve of PKA

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  < Real-Time Assay >
  
• 8. Real-time Assay Protocol
  
• 9. Performance
  
  
  • 9-1. Comparison of ADP Real-time quantification time
  • 9-2. Effect of addition of DTT at real-time assay
• 10. Data: Calculation of Km (ATP) of PKA by the real-time assay

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• 11. LINK
  • Leaflet (1 MB/4 p)
  • Package Insert (1,7 MB/20p)

1. Kumagai, K. et al.: Anal. Biochem., 447, 146-155 (2014).
2. Drug Discovery Initiative, The University of Tokyo http://www.ddi.u-tokyo.ac.jp/en

6-1. Preparation of Standard Curve of ADP

Prepare the standard curve using 0, 10, 20 and 30μmol/L ADP.

The result indicated that the standard curve keeps linearity up to 30 μmol/L of ADP.

6-2. ADP Standard Curve at the low substrate conversion rate

Prepare the several standard curve with the substrate conversion rate is 0 ~ 10% at ATP and ATP concentration is 1, 10 and 100 μmol/L.

The result indicated that the quantitative capability is successful in the condition
of the low substrate conversion rate (= the low concentration of ADP)

6-3. Fluorescent signal stability

20 μmol/L ADP and 2 x Detection solution were mixed and after 30 minutes to 7 hours the fluorescent intensity was measured. After 30 minutes, the fluorescence intensity was standardized at 100% and the variability plotted on a graph.

The fluorescence intensity was stabilized during the 30 minute to 7 hour interval
after ADP and 2 x Detection Solution were mixed.

7-1. Calculation of Z'-factor

Z' -factor was calculated at 5% Conversion Rate (ADP/ATP = 1, 10 and 100 μmol/L each)（ATP+ADP= 1, 10, 100μmol/L). The corresponding 5% substrate conversion rate of ADP was quantified and the Z'-factors of this technique and conventional spectroscopic methods were compared.

Z'-factor : Fluorospark^™ Kinase > Conventional bioluminescent method (Company A)
Superior Z'-factor values can be acquired even for low-substrate conversion rates of 5% with regards to the various concentrations of ATP.

Z'-factor is ...

  Z'-factor is one of the static values to evaluate the performance of the HTS assay in terms of sensitivity and accuracy. 
      The Z’-factor > 0.5 indicates the robust assay.
      Z’-factor = 1 - (3 × SDH + 3 × SDL)/(AvH - AvL)
         ⇒ SDH, SDL: Standard deviation of high and low control group, respectively
         ⇒ AvH, AvL: Mean value of high and low control group, respectively
      The nearer 1 the Z’-factor is, the more sensitive and lower variable assay system this kit is.

7-2. Inhibition Curve of PKA

Preparation of the inhibition curve of cAMP-dependent protein kinase(PKA) by PKA specific inhibitor, H-89.
(PKA Catalytic Subunit 0.1 U, ATP 5 μmol/L, Kemptide 125 μg/mL）

IC50 with Fluorospark™ Kinase/ADP Multi-Assay Kit is nearly equivalent to that with a conventional method (Company A)

The result indicated that the determined IC₅₀ value was consistent with the reference's value* (IC₅₀ = 40 nmol/L

* Hidaka, H. et al., Methods Enzymol., 201, 328-39 (1991).

9-1. Comparison of ADP Real-time quantification time

A real-time assay was performed with various concentrations of ADP [ADP = 0, 0.25, 0.5, 1, 2 μmol/L (ATP + ADP = 10 μmol/L)]

Quantitative ADP reaction were completed within approximately 10 minutes
with the Fluorospark™ Kinase assay with a stable background signal (ADP = 0 μmol/L).

9-2. Influence of the addition of DTT in the Real-time Assay

A Real-time assay was performed with the addition of the reducing agent DTT (0.1, 1 mmol/L) with ADP [2 μmol/L (ATP + ADP = 10 μmol/L).

Quantitative ADP reactions were complete within approximately 10 minutes with the Fluorospark™ Kinase assay.
Stable results were acquired even in the presence of high concentrations of DTT.