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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


  • Applicable to Endpoint & Real-time assay, respectively
  • Excellent Z'-factor at low substrate conversion rate
  • High Sensitive
  • Standard curve keeps the linearity up to 30 μmol/L of ADP.
  • 30 minute reaction with one-step protocol
  • Lower cost
References
  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

2. Comparison with conventional (luminescent) method


  Fluorospark Kinase/ADP Multi-assay Kit Conventional luminescent method
(Company A)
Principle
ADP determination (Fluorescent)
(Generated ADP is directly determined)
ADP determination (Luminescent)
(Converted from generated ADP to ATP and determined the ATP)
Pipetting step
1 step
2 steps
Operation time
30 minutes
70 ~ 100 minute
ADP measuring range
~ 30 μmol/L
~ 1 mmol/L
Price
Lower
Higher
Advantage
High Z'-factor
Applicable to Real-time-Assay
Wide dynamic range
Higher S/B ratio

3. Summary


Summary


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.

4. Kit Contents


( No. )        Reagent               Intended purpose 1,000 tests 10,000 tests
(1) Substrate Solution
Preparation of 2 x Detection Solution
9 mL
90 mL
(2) Enzyme Solution
500 μL
5 mL
(3) Resazurin Solution
100 μL
1 mL
(4) Reductant Blocker
400 μL
4 mL
(5) Stop Solution
for stopping of the enzymatic coupling reaction
10 mL
100 mL
(6) 10 mmol/L ATP Solution
for kinase reaction and preparing a standard curve
100 μL
1 mL
(7) 10 mmol/L ADP Solution
for preparing a standard curve
100 μL
1 mL

< Endpoint assay >

5. Protocol of Endpoint assay



PROTOCOL


Kinase activity can be measured by preparing and adding 2 x Detection Solution.

 


6. Performance


6-1. Preparation of Standard Curve of ADP

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


Result

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.


Data

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.


Result

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


7. Data



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.


Calculation of Z'-factor

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)


Inhibition Curve

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).

< Real-time Assay >

8. Real-time Assay Protocol


Measurement

Kinase activity can be determined by measuring the fluorescence continuously.

9. Performance


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)]


real-time_ADP

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).


Reatl-time DTT

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.

10. Data: Calculation of Km (ATP) of PKA by the real-time assay


(Add 50 μL of kinase reaction solution (including PKA 0.02 U/μL, the several concentration of ATP and Kempeptide 100 μg/mL)




measurement

Using this real-time assay, enzyme-based kinetic analyses are feasible.
Km values obtained using this technique were approximately equal to those found in the literature (3-15 μmol/L).

** Flockhart, D.A. and Corbin, J.D., CRC Crit. Rev. Biochem., 12, 133-86(1982).



Product Name Grade Package Size Wako Cat. No. Storage Condition
Fluorospark Kinase/ADP Multi-Assay Kit
for Enzyme Activity Assay
1,000 tests
Keep at -20°C.
10,000 tests

  • Since clicking on the product code number will take you to an e-reagent.com link, please confirm the current price there.
  • The number of uses of this kit is based on the usage of a full-volume 384-well plate. In the event that other microwell-plates are to be used, the numbers of usage of this kit may vary.
  • When using a low-volume 384-well plate, the Fluorospark™ Kinase/ADP Multi-Assay Kit (Wako Cat. No. 291-77401 (1,000 tests)) can be used 2,000 times.
  • When using a 96-well plate, Wako Cat. No. 291-77401 (1,000 tests) can be used 200 times.





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