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.
![]() < Endpoint assay > |
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
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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
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
Kinase activity can be measured by preparing and adding 2 x Detection Solution.
6. Performance
7. Data
< Real-time Assay >
8. Real-time Assay Protocol
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Kinase activity can be determined by measuring the fluorescence continuously.
9. Performance
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)
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 |
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[License] Fluorospark® is the registered trademark of Wako in Japan. |