激酶活性检测试剂盒R&D

Universal Kinase Activity Kit

Catalog Number: EA004

The protocol presented is for a 96-well format. Materials

provided are sufficient for two 96-well microplates or

equivalent. This package insert must be read in its entirety

before using this product.

ASSAY PRINCIPLE

The Universal Kinase Activity Kit provides a convenient,

non-radioactive high-throughput compatible format for

assaying all kinases. This kit takes advantage of a

phosphatase that removes inorganic phosphate

quantitatively from ADP. The released phosphate is then

detected with malachite green phosphate detection

reagents (Figure 1). The amount of inorganic phosphate

released by the coupling phosphatase is proportional to

the ADP generated; therefore, the rate of inorganic

phosphate production reflects the kinetics of a kinase

reaction (1).

Figure 1: Kinase activity assay kinase

reaction converts ATP to ADP. The coupling phosphatase,

CD39L2, releases one inorganic phosphate from ADP. The

inorganic phosphate is detected using malachite green

phosphate detection reagents.

MATERIALS PROVIDED

Upon receipt, store ATP, ADP, and Coupling

Phosphatase 4 at£-20 °C until use. All other

components may be stored at ambient temperature.

Use before the kit expiration date.

Coupling Phosphatase 4(Part 895988, 1 vial)- CHO

cell-expressed recombinant mouse CD39L2/ENTPD6;

200mL of 100 ng/mL in 25 mM Tris, 150 mM NaCl, 5 mM

CaCl

2

, 20% glycerol, pH 7.5.

Phosphatase Buffer 4(Part 895989, 2 vials)- Each vial

contains 1.5 mL of a 10X solution of 250 mM HEPES,

1.5 M NaCl, 100 mM MgCl

2

, 100 mM CaCl

2

, pH 7.0.

ATP(Part 895990, 2 vials)- Each vial contains 200

L of

10 mM ATP in 25 mM sodium borate, pH 9.0.

ADP

(Part 895991, 1 vial)- 200mL of 10 mM ADP in

25 mM sodium borate, pH 9.0.

Phosphate Standard(Part 892809, 1 vial)- 200mL of

1 mM phosphate (KH

2

PO

4

) in deionized water.

Malachite Green Reagent A(Part 895855, 3 vials)-

Each vial contains 3 mL of ammonium molybdate in 3 M

sulfuric acid.

Malachite Green Reagent B(Part 895856, 3 vials)-

Each vial contains 3 mL of malachite green oxalate and

polyvinyl alcohol.

MATERIALS REQUIRED

·

·

·

·

·

Kinase.

Acceptor substrate for kinase.

Assay buffer (if different than provided).

Deionized or distilled water.

Microplate reader capable of measuring absorbance

at 620 nm.

·

Microplate (R&D Systems, Catalog # DY990 or

equivalent).

PRECAUTION

Malachite Green Reagent A, Malachite Green Reagent B,

and Phosphate Standard supplied with this kit are acidic

solutions. Wear protective gloves, clothing, eye, and face

protection. Wash hands thoroughly after handling.

REAGENT PREPARATION

The volumes of working solutions below are for 20 kinase

reactions. Adjust the volumes accordingly if a different

number of reactions is planned. Discard the leftovers of

the preparations after the assay is complete.

1X Assay Buffer- Add 200mL of 10X Phosphatase

Assay Buffer 4 to 1.8 mL of deionized or distilled water in

a tube. Mix well.

Prepare the following items in the 1X Assay Buffer.

·

Kinase: 300mL at 1 ng/mL to 1mg/mL

·

Acceptor Substrate: 300mL at 0.5-5 mM

·

ADP: 100

L at 1 mM

·

ATP: 100

L at 1 mM

·

Coupling Phosphatase 4: 200mL at 10 ng/

L

PHOSPHATE STANDARD CURVE

DETERMINATION

This protocol is for determining a standard curve in a

96-well microplate. It is recommended that the standards

be assayed in duplicate and a standard curve be

generated before an enzyme kinetic assay.

40mL of the 1 mM Phosphate Standard to 360mL

of 1X Assay Buffer in a microcentrifuge tube and mix

well. Transfer 200mL of the dilution into 200mL of

1X Assay Buffer in a second tube. Repeat the process

to prepare a 2-fold serial dilution. The eighth tube

contains only 1X Assay Buffer and serves as the

blank.

er 50

L of each dilution into microplate wells in

duplicate.

30mL of Malachite Green Reagent A to each

well. Mix by gently tapping the plate.

100mL of deionized or distilled water to each

well.

30mL of Malachite Green Reagent B to each

well. Mix by gently tapping the plate.

te the plate for 20 minutes at room temperature

to stabilize the color development. The yellow

background will fade during incubation.

ine the optical density (OD) of each well using

a microplate reader set to 620 nm.

e the duplicate readings for each standard and

subtract the OD of the average zero standard. Plot

phosphate input (pmol) vs. the corrected OD

(Figure 2).

Figure 2:A phosphate standard curve determined

using 1X Assay slope of the linear

regression line, 3504.3 pmol/OD, represents the amount

of phosphate corresponding to a unit of absorbance at

620 nm. It is referred to as the phosphate conversion

factor (CF) in subsequent calculations. CF may vary

under different conditions. This standard curve is provided

for demonstration only.

KINASE ASSAY PROTOCOL

This standard kinase assay is carried out in 50mL for

10 minutes at room temperature and is coupled to 100 ng

of Coupling Phosphatase 4. The reaction may be scaled

up proportionally. 1X Assay Buffer can be used in most

kinase reactions. Individual kinase reactions can be

optimized by adjusting the concentrations of kinase,

substrates, and metal ions. It is recommended that all

samples be assayed in duplicate. If assay conditions

change, the amount of coupling enzyme should be

adjusted to reach the optimal coupling rate. For more

information, see the Technical Hints and Limitations

section.

e the substrate mix using the freshly prepared

working solutions.

ATP10mL

Acceptor15mL

Total Volume25mL

2.

Prepare the enzyme mix using the freshly prepared

working solutions.

Coupling Phosphatase 410mL

Kinase

15mL

Total Volume25mL

3.

For a positive control, use ADP in place of ATP.

a negative control, use 1X Assay Buffer in place of

the kinase.

5.

Initiate the reaction by adding the substrate mix to the

enzyme mix in a 96-well microplate.

6.

As an assay blank, add 50mL of 1X Assay Buffer in a

separate well.

7.

Incubate the microplate for 10 minutes at room

temperature.

ate the reactions by adding 30L* of Malachite

Green Reagent A to each well. Mix by gently tapping

the microplate.

9.

Add 100

L* of deionized or distilled water to each

well. Mix gently by tapping the microplate.

10.

Add 30L* of Malachite Green Reagent B to each

well. Mix gently by tapping the microplate.

te the microplate for 20 minutes at room

temperature to stabilize the color development.

ine the optical density of each well using a

microplate reader set to 620 nm, and adjust the OD by

subtracting the negative control.

ate the phosphate in each reaction using the

previously determined conversion factor.

*If altering the reaction volume, it is necessary to maintain

a ratio of the reaction volume plus water : Malachite

Green Reagent A : Malachite Green Reagent B of 5:1:1.

ASSAY EXAMPLE

Figure 3: PKA inant human PKA Cb

(R&D Systems, Catalog # 4596-KS) was assayed with

0.2 mM ATP, 0.4 mM Kemptide (,

Catalog # 22595), and 0.1mg of Coupling Phosphatase 4

in 50mL of 1X Assay Buffer for 10 minutes at room

temperature. When the corrected optical density was

plotted versus the amount of kinase, a slope of

0.908 OD/mg was obtained. Using the CF of

3504 pmol/OD determined in Figure 2 and the equation

(specific activity = slope x (CF/time)), the specific activity

was calculated to be 318 pmol/min/mg. After correction

with the coupling rate, 0.475 (Table 1), the activity is

670 pmol/min/mg.

TECHNICAL HINTS AND LIMITATIONS

·

Malachite Green Reagents are highly sensitive to

phosphate. All reagents must be phosphate-free. In

particular,phosphate-containing buffers should be

avoided at all a phosphate-containing enzyme

preparation is to be assayed, its phosphate content

should be removed using dialysis or chromatography

steps.

The linear response region for phosphate detection is

between 100-4000 pmol. Higher levels of phosphate may

cause precipitation of the phosphate-malachite complex.

To ensure that the phosphate content falls into this range,

a portion of the reactions may be used for detection.

Alternatively, the reactions can be diluted prior to

detection. In any case, the ratio of reaction plus water :

Malachite Green Reagent A : Malachite Green Reagent B

should be kept at 5:1:1.

Although CD39L2 prefers ADP as a substrate, it has

slight activity on ATP. Increasing the amount of coupling

phosphatase will increase the hydrolysis of ATP,

therefore resulting in higher reduce the

background while maintaining a good signal, the

amount of coupling phosphatase needs to be

restricted to achieve an optimal coupling rate

between 0.4 and 0.6.A coupling rate is defined as the

ratio of the product ADP that has been converted to the

signal of free inorganic phosphate (1). The coupling rate

of a kinase reaction is used to calculate the amount of

ADP produced by the kinase reaction based on the

amount of phosphate detected. Coupling rates for assays

under different ATP concentrations using the provided

protocol are listed in Table 1.

The coupling rate is pH, NaCl, ATP, and temperature

2+

sensitive and Cadependent (1). If these conditions are

changed, the coupling rate should be recalculated, which

further requires the determination of the rate constant of

the coupling enzyme. For rate constant determination and

coupling rate calculation, see reference 1 or the technical

information available on .

ATP is less stable under the acidic conditions of

Malachite Green Reagents, which will result in slowly

increasing background with time. For consistency, all

reactions should be read 20 minutes after the addition of

Malachite Green Reagents.

If the reaction conditions for a kinase and the coupling

phosphatase are not compatible, a decoupled assay is

suggested, in which phosphatase and phosphatase

buffer are added into the kinase reaction for an additional

10-20 minutes. In this case, the strength of the

phosphatase buffer is recommended to be 4X higher than

that of the kinase buffer.

A decoupled assay is also recommended for kinases that

have low activities or require reaction time of more than

30 minutes to avoid high background from ATP hydrolysis

by CD39L2.

DTT may be required for a protein kinase assay and can

be added into a kinase reaction at 1 mM without adverse

effect to the coupling enzyme and the phosphate

detection.

Pipetting concentrated proteins or polypeptides can

cause foaming, which should be eliminated before

measurement.

·

·

·

·

·

·

·

·

Table 1: Coupling rates for 10 minute 50mL reactions

using the supplied buffer at room temperature.

Coupling rates under different ATP concentations and

varying amounts of CD39L2 are listed. The coupling rate

is a function of the rate constant, reaction volume, and

reaction time. Calculations are based on the specific rate

constant of 97.78 nmol/min/mg/mM. For best performance,

coupling rates are suggested to be between 0.4 and 0.6.

For calculating coupling rates under different assay

conditions, such as incubation time or the amount of

coupling enzyme, refer to reference 1 or technical

information available at .

ATP

(mM)

100

200

300

400

500

600

700

800

900

1000

CD39L2 (

g)

0.050

0.309

0.291

0.279

0.269

0.262

0.255

0.249

0.244

0.239

0.234

0.075

0.415

0.394

0.379

0.367

0.357

0.349

0.342

0.335

0.329

0.324

0.100

0.498

0.475

0.460

0.447

0.436

0.427

0.419

0.411

0.405

0.399

0.125

0.564

0.542

0.525

0.512

0.501

0.492

0.483

0.476

0.468

0.462

0.150

0.618

0.596

0.580

0.567

0.556

0.546

0.537

0.530

0.522

0.516

0.175

0.662

0.640

0.625

0.612

0.601

0.592

0.583

0.576

0.568

0.562

0.200

0.697

0.677

0.662

0.650

0.640

0.631

0.622

0.615

0.608

0.601

REFERENCE

, Z.L. (2011) PLoS ONE6(8):e23172.

APPENDIX

The following detergents and common reagents were

tested for interference in the phosphate assay. The effects

occurred at concentrations above those listed.

Detergents

1

Triton

Ò

X-100

Tween

Ò

20

NP-40 Alternative

CHAPS

SDS

Deoxycholate

Common Reagents

1

Glycerol

DMSO

Ethanol

BSA

EDTA

Dithiothreitol

b-mercaptoethanol

Na

3

VO

4

NaF

NaCl

KCl

CaCl

2

1

Level

0.3%

0.1%

1%

1%

0.01%

0.01%

Effect

Increased Blank

Reduced Sensitivity

None

None

Increased Blank

Increased Blank

Precipitates

Effect

Reduced Sensitivity

Reduced Sensitivity

Reduced Sensitivity

Reduced Sensitivity

None

Reduced Sensitivity

None

Reduced Sensitivity

None

None

None

None

Level

5%

10%

25%

0.03 mg/mL

10 mM

3 mM

10 mM

1 mM

10 mM

100 mM

100 mM

10 mM

Tested using the microplate assay protocol in 25 mM Tris-HCl,

pH 7.5, with or without 1 nmol phosphate (KH

2

PO

4

).

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respective owners.

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

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