CatchPoint Cyclic-GMP Fluorescent Assay Kit产品说明书

Product Insert

CatchPoint

™

Cyclic-GMP Fluorescent Assay Kit

Product # R8075

(Bulk Kit)

Quantity: 960, 120 µL reactions

Introduction

About the cGMP Assay

The CatchPoint

™

cyclic-GMP Fluorescent Assay Kit measures levels of 3’, 5’-cyclic

guanosine monophosphate (cGMP) or guanylate cyclase activity via a competitive

immunoassay for cGMP. The assay requires only a single washing step, and readings can

be taken in as little as 10 minutes or as long as 24 hours following substrate addition,

since no termination step is needed. The cGMP Fluorescent Assay Kit is suitable for use

in cell-based assays.

Principle of the Assay

The cGMP in the sample or standard competes with horseradish peroxidase (HRP)-

labeled cGMP conjugate for binding sites on the anti-cGMP antibodies (Fig. 1). In the

absence of cGMP, most of the HRP-cGMP conjugate is bound to the antibody. Increasing

concentrations of cGMP competitively decrease the amount of bound conjugate, thus

decreasing measured HRP activity.

HRP

HRP HRPHRP

HRPHRP

HRP

No cGMP Increasing cGMP

Maximum HRP activity

Decreasing HRP activity

cGMP

cGMP-HRP

HRP

Conjugate

Goat anti-Rabbit IgG

Rabbit anti-cGMP coated microplate

Antibody

Figure 1. Principle of the CatchPoint cGMP assay system

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Applications

The kit is designed for use in applications where cGMP is generated in biochemical assay

systems, such as assays of guanylate cyclase activity. In addition, this kit can be used for

cell-based assays to measure intracellular cGMP levels following stimulation with peptide

hormones or stimulants such as nitric oxide (NO), sodium nitrate, and nitroprusside to

soluble or particulate guanylate cyclases.

Kit Components

Materials and Equipment

The following table lists the kit components.

Table 1: The CatchPoint cGMP Fluorescent Assay Kit (P/N R8075) contents

Reagent Quantity Description

96-well black plates 10 plates 96-well plate coated with Goat anti-

Rabbit IgG. Plates are pre-blocked and (clear bottom)

ready to use.

cGMP Assay Buffer 950 mL

Rabbit anti-cGMP Antibody 2 vials Antibody, lyophilized

cGMP Calibrator 2 vials

30 µM cGMP Calibrator, lyophilized.

On reconstitution with 5 mL assay

buffer each vial contains 30,000 pmol

cGMP / mL.

HRP-cGMP Conjugate 2 vials HRP-cGMP Conjugate, lyophilized

10X Wash Concentrate 950 mL

Cell Lysis Buffer 450 mL

Stoplight Red  Substrate 2 x 900 µL

100X Substrate in DMSO

Substrate Buffer 950 mL

When the working concentrations of the above reagents are used as suggested, each kit

provides sufficient reagents for ten 96-well microplates with 120 µL total assay volume.

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Storage and

All kit components are to be stored at 4°C.

Handling

IMPORTANT: Allow all the reagents to warm to room temperature prior to use.

Note: Small volumes of product will occasionally collect in the cap of the product vial during

shipment. Gently tap the vial on a hard surface or briefly centrifuge the vial to collect any liquid

trapped in the vial cap.

When stored properly, the kit components are stable for six months from the date of receipt.

The reconstituted working solutions of Rabbit anti-cGMP, cGMP Calibrator, and HRP-cGMP

Conjugate are stable for 3 weeks at 4°C. To insure optimal performance of the reagents do not

store below 4°C once reconstituted.

If the entire plate will not be used, protect any unused wells with a plate sealer and store at 4°C

in the original foil wrapper protected from light.

IMPORTANT: The reconstituted substrate solution is sensitive to light. To insure optimal

performance we recommend preparing a fresh stock solution (keep protected from light) and

adding to the assay plate within 60 minutes. Following substrate addition readings can be

taken at 10 minutes or as long as 24 hours.

Materials

Required but

not Provided

The following tables list the materials required but not supplied.

Table 2: Reagents and supplies

Reagent Item

Source

3% wt / vol hydrogen peroxide (H

2

O

2

) Major laboratory suppliers (MLS)

MLS

MLS

MLS

1.5 mL polypropylene capped

centrifuge tubes

10 mL capped centrifuge tubes

Pipettors – adjustable volume

Multi-channel troughs MLS

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Equipment Item

Table 3: Compatible Instruments available from Molecular Devices (MDC)

Source

Analyst System

One of the following:

- Analyst GT

- Analyst HT

MDC P/N 0200-6003 or 0200-6004

MDC P/N 0200-6043 or 0200-6044

Gemini XPS

MDC P/N Gemini XPS Spectrophotometer

Gemini EM

MDC P/N Gemini EM Spectrofluorometer

SpectraMax M2

SpectraMax M5

FlexStation

®

®

MDC P/N M2

MDC P/N M5

MDC P/N FlexStationII Scanning Fluorometer

Embla 96/384 plate washer

MDC P/N 0200-3948

AquaMax DW4 with 96-well

MDC P/N AquaMax DW4 Liquid Handling System

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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CatchPoint cGMP Fluorescent Assay Kit Experimental

Protocol

Preparing the

Reagents

Table 4: Preparation of reagents

Step Action

1

Reconstitute one vial of 30 µM cGMP Calibrator:

Add 5 mL of cGMP Assay Buffer to the vial. Mix well

to ensure dissolution of all contents. Store on ice or at 4°C.

2 Reconstitute one vial of HRP-cGMP Conjugate:

Add 10 mL of cGMP Assay Buffer to one of the vials for a 10X stock.

Mix well to ensure dissolution of all contents. Transfer the entire

contents of the vial to a 100 mL graduated cylinder by repeated

washings with assay buffer. Adjust the final volume to 100 mL with

assay buffer and mix thoroughly. The diluted HRP-cGMP conjugate is

ready for use in the assay. Store on ice or at 4°C.

3 Reconstitute one vial of Rabbit anti-cGMP Antibody:

Add 10 mL of cGMP Assay Buffer to one of the vials for a 10X stock.

Mix well to ensure dissolution of all contents. Transfer the entire

contents of the vial to a 100 mL graduated cylinder by repeated

washings with assay buffer. Adjust the final volume to 100 mL with

assay buffer and mix thoroughly. The diluted Rabbit anti-cGMP

conjugate is ready for use in the assay. Store on ice or at 4°C.

4 Dilute 10X Wash Concentrate 10-fold in deionized

water, i.e. for one assay plate dilute 500 mL of 10X Wash Concentrate

in 4500 mL of deionized water.*

5

Prepare calibrators: The 30 µM cGMP calibrator is diluted in cGMP

Assay Buffer to prepare stock calibrators of 10000, 100, 33, 11, 3.7,

1.2, and 0.4 nM cGMP. These give 3300, 33, 11, 3.7, 1.2, 0.41, and

0.14 nM (400, 4.0, 1.3, 0.44, 0.15, 0.049, and 0.016 pmol) final

concentrations in the assay.

1. Add 500 µL of the 30 µM cGMP calibrator to 1000 µL of cGMP

Assay Buffer. This is the 10,000 nM stock calibrator.

2. Add 20 µL of the 10,000 nM stock calibrator to 1980 µL of cGMP

Assay Buffer. This is the 100 nM stock calibrator.

3. In cGMP Assay Buffer, serially dilute the 100 nM stock calibrator in

a 3-fold fashion five times to produce the 33, 11, 3.7, 1.2, and 0.4

nM cGMP stock calibrators. 40 µL of each calibrator are required

per replicate. Prepare 1000 µL of each (ample for 10 replicates).

As a 0 nM (zero dose) calibrator, use the cGMP Assay Buffer itself.

The following protocol provides sufficient reagents to run five 96-well plates, each with 16

calibrators, 2 controls, and up to 78 samples.

* On dilution of the 10X Wash Buffer concentrate, the reagent contains 0.02 M Tris, 150 mM

NaCl, 0.05% Tween 20, and 0.05% Proclin 200 (pH 7.4).

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Preparing the

Plate

For ease of analysis and calculation, the assay plate contains the calibrators, controls,

and samples as outlined below. Use the suggested template (Table 5) and run the

samples in duplicate.

Note: The order of addition of each component to the wells and the incubation time are

important. Mix samples and all reagents gently but thoroughly before use.

All assay wells will be prepared by dispensing the assay components in the following order:

1) Calibrator, control, or sample

2) Rabbit anti-cGMP Antibody

3) HRP-cGMP Conjugate

Table 5: To prepare the assay plate:

Step Action

Designing your assay plate (plate map):

1

We suggest preparing the plate according to the template below. If you

are using SoftMax® Pro to analyze your results, you may set up a

template before or after reading the plate. If you set up the plate before,

you can print out a template to help in preparing your plate.

Column

Row1 2 3

A 3300 nM 3300 nM Control 1

(400 pmol) (400 pmol)

B 33 nM 33 nM Control 2

(4 pmol) (4 pmol)

C 11 nM 11 nM

(1.3 pmol) (1.3 pmol)

D 3.7 nM 3.7 nM

(0.44 pmol) (0.44 pmol)

E 1.2 nM 1.2 nM Column 3 (C –H) and

(0.15 nM) (0.15 nM) all of columns 4 - 12:

F 0.41 nM 0.41 nM Samples for analysis

(0.049 pmol) (0.049 pmol)

G 0.14 nM 0.14 nM

(0.016 pmol) (0.016 pmol)

H 0 nM 0 nM

(buffer only) (buffer only)

2

Add 40 µL of the appropriate concentration of Calibrator working

solution (see step 5 from “Preparing the Reagents” on Table 4, page 4)

to columns 1 and 2 (rows A – H). Column 3 (A & B) is designated here

for optional controls. For example, one may wish to use a zero dose

calibrator with no Rabbit anti-cGMP Antibody to evaluate background.

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Table 5: To prepare the assay plate:

3

Place 40 µL of samples to be analyzed in appropriate wells [e.g.

Column 3 (C – H), and columns 4 – 12]. See Appendix A for cell

stimulation and lysis protocols.

4

Add 40 µL of reconstituted Rabbit anti-cGMP Antibody to all wells

except any reserved for no antibody controls. If these are to be

performed, add 40 µL assay buffer in place of antibody.

Place plate on shaker for 5 minutes or gently agitate by hand to ensure

5

mixing.

6

Add 40 µL reconstituted HRP-cGMP Conjugate to every well.

Mix well and allow to incubate 2 hours at room temperature.

7

8

Aspirate plate contents and wash 4 times with 300 µL wash buffer

for each wash.

9

Prepare Stoplight Red Substrate: Dilute 600 µL of 100X stock Stoplight

Red Substrate into 60 mL of Substrate Buffer, then add 68 µL of 3%

H

2

0

2

(880 mM) to bring the final concentration to 0.0034% (1 mM) H

2

0

2

.

Important: The reconstituted substrate solution is sensitive to light. To

insure optimal performance we recommend preparing a fresh stock

solution and adding directly to assay plate within 60 min. At all times

keep substrate protected from light.

10

Add 100 µL Stoplight Red substrate to every well, minimizing the time

between starting and finishing. Cover the plate and leave at room

temperature for at least 10 minutes, shielded from light.

Read the fluorescence intensity of the plate on an appropriate

11

instrument (see below for settings). Plates may generally be read

anytime between 10 minutes and 24 hours. For optimal performance

you may want to briefly mix the plate prior to reading.

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Analyst Parameter Reading the Plate in Analyst HTS Assay Detection System

Settings

The following instrument settings are recommended when using 96-well plates with a

100 µL volume in Analyst HTS systems:

Table 6: Fluorescence intensity settings for Analyst HT or GT Detection System:

Parameter

Setting

Fluorescence Intensity

530-25 nm

590-20 nm

Mode

Excitation filters

Emission filters

Dichroic mirror 50 / 50 beamsplitter

Z-height 3 mm

Attenuator Medium

Integration time

50,000 µsec

Lamp Continuous

Readings per well

PMT setup

Units

One

Smart Read+ (sensitivity = 2)

Counts / sec

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Reading the Plate in the Gemini XS, Gemini XPS, Gemini EM, SpectraMax

®

M2 and

SpectraMax

®

M5 Detection System

The following instrument settings are recommended when using 96-well plates with a

Gemini XS system:

Table 7: Fluorescence intensity settings for GEMINI XS, Gemini XPS, Gemini EM,

SpectraMax

®

M2 and SpectraMax

®

M5 Detection System

Parameter Setting

Read Mode

Endpoint

Fluorescence (RFUs)

530 nm

590 nm

570 nm

6

Auto

On

Read Type

Excitation

Emission

Cutoff

Sensitivity

PMT

AutoCalibrate

Data Analysis

Analyzing the Calibration Data

The average intensity values (y-axis) can be plotted against calibrator concentration (x-axis)

to create a calibration or standard curve. Unknown samples can be interpolated using this

curve to calculate cGMP doses in the samples. We recommend using SoftMax Pro software

for accurate curve fitting and interpolation of data from MDC instruments. Figure 2 shows a

SoftMax Pro template and calibration curve for the cGMP assay when read on the Analyst™.

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Figure 2. Calibration curve for the CatchPoint cyclic GMP Fluorescent Assay Kit, read on the Analyst system.

Data was taken 60 minutes after addition of Stoplight Red substrate. Fluorescence intensity settings for Analyst

HTS Detection System were as stated in Table 6. The EC

50

value of the calibration curve, calculated as

coefficient C using SoftMax Pro, was 2.2 nM (264 fmol).

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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ADDITIONAL INFORMATION

The following typical information is based on data from an Analyst HT. Absolute readings may differ on other

instruments, but relative values should be comparable.

Sensitivity (Typical values)

Minimum Detectable Concentration = 0.2 nM (24 fmol / well)

Maximum Measurable Concentration = 120 nM (14 pmol / well)

EC

50

= 2.5 + 0.6 nM (300 fmol)

Intra-assay precision

Intra-assay precision was calculated from measurements of 0 and 1.2 nM calibrator. The results

for a single assay are shown below:

Signal intensity / background

(for 0 nM calibrator)

§

%B/B

o

* precision for 1.2 nM calibrator

Signal intensity

Avg

/ background = 290

Standard deviation (SD) = 12

%CV = 4

n = 23

%B/B

o

(mean) = 59

Standard deviation (SD) = 4

%CV = 7

n = 23

§

Background taken as the signal intensity for wells in which no cGMP-HRP or Rabbit anti-cGMP

Antibody are added

* %B/B

o

= 100 X (I

1.2 nM

– I

∞

) / (I

o

– I

∞

)

where I =

1.2 nM

I =

o

I

=

∞

Signal intensity for 1.2 nM calibrator

Signal intensity for 0 nM calibrator

Signal intensity for

3333 nM calibrator

Inter-assay precision (three assays performed on separate days)

Inter-assay precision was calculated from measurements of %B/B

o

for 1.2 nM calibrator in 3 successive assays.

The results are shown below:

Measurement

Data points

%B/B

0

58

61

59

SD

5

4

4

%CV

8

6

7

A

B

C

n = 2

n = 2

n = 23

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Precision correlation

22 replicates of each of the standards were prepared and % coefficient of variation was determined for each

concentration using SoftMax Pro.

Sample Concentration Mean Value

SD %CV

cal 01 3300 nM

2084707826

3.8

cal 02 33 nM

29

6.8

cal 03 11 nM

4876559202898

4.2

cal 04 3.7 nM

18

4.4

cal 05 1.2 nM

17

4.7

cal 06 0.41 nM

22

4.5

cal 07 0.14 nM

243627261233519

5.1

cal 08 0 nM

285929641049992

3.7

Appendix A: Preparing cell lysates for the cGMP Assay

The protocol below illustrates an application of the CatchPoint cGMP Fluorescent Assay. The protocol has

been developed for use with adherent cells stimulated with atrial natriuretic peptide (ANP). The procedure may

be modified for various hormones and stimulators, such as acetylcholine, insulin, serotonin, and nitric oxide.

For best results each cell line should be evaluated to determine the optimal concentration of cells per well,

incubation times, and concentration of guanylate cyclase agonists or antagonists.

Use this protocol as a guide to help optimize the assay.

______________________________________________________________________________________

Reagents

Reagent Stock Concentration

cGMP stimulator atrial natriuretic peptide,

300 µM

dissolved in phosphate buffered saline (PBS)

Phosphodiesterase inhibitor, 3-isobutyl-1-800 mM

methylxanthine (IBMX) dissolved in DMSO

Buffers

Buffer Source

Krebs-Ringer Bicarbonate Buffer (KRBG) Sigma Chemical Company (P/N K4002)

containing 10 mM Glucose (pH 7.4)

Note: 15 mM sodium bicarbonate is not

included with this product and must be

added separately.

Lysis Buffer (pH 7.3) Molecular Devices Corporation (included in

CatchPoint cGMP Fluorescent Assay Kit)

Phosphate Buffered Saline, PBS

Invitrogen Life Technologies

(P/N 10010-023)

CatchPoint Cyclic-GMP Bulk Fluorescent Assay Kit_96-well format

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Assay Protocol

Adherent Cells

The following has been used for the adherent cell lines RFL-6 and CHO-K1 :

1) Culture cells (1 mL / well) in standard 12-well microtiter plates (tissue culture grade) with cell concentration

at 0.75 – 2.5 x 10

5

cells/mL.

2) Incubate plated cells overnight at 37°C in a humidified atmosphere of 5% CO

2

: 95% air.

3) Gently aspirate media and slowly add 400 µL of Pre-stimulation Buffer (pH 7.4).

Pre-stimulation Buffer (containing 0.75 mM IBMX in KRBG Buffer; make fresh on day of experiment)

10 ml of KRBG Buffer (pH 7.4)

9.4 µL of 800 mM IBMX

Note: Once IBMX has been added to the KRBG, mix vigorously to obtain a homogeneous solution.

4) Incubate for 10 min at room temperature.

5) Add 200 µL of 3X ANP or PBS (negative control). Gently mix and incubate at 37°C for 15 minutes.

ANP, 3X (500 nM final concentration)

2.0 mL PBS (pH 7.4) containing

10 µL of 300 µM ANP (1.5 µM stock)

6) Add 200 µL of lysis buffer to each well. Agitate cells to facilitate cell lysis. This can be achieved by shaking

the plate on a plate shaker for 10 min after adding the lysis reagent.

7) If desired, carry out microscopic evaluation using Trypan Blue to check if cells have lysed (1:1 ratio). Cell

membrane may still be visible after cell lysis. Lysed cells are now ready for use and should be immediately

processed in the cGMP Fluorescent Assay Kit. Use 40 µL per well neat or suitably diluted lysate in

additional lysis buffer (see page 6, Table 5, Step 3 of the Product Insert).

8) Proceed with the remainder of the procedure in Table 5.

Cells in Suspension

For non-adherent cells, we recommend centrifugation of the cells from culture medium and suspension of the

pellet in Pre-stimulation Buffer (pH 7.4). Add 1 mL (12 well plate) of cell suspension to each well of the plate.

It is recommended that you then centrifuge the plates at 1000 rpm for up to 4 min (brake off). Proceed with step

3 (shown above) for adherent cells.

R3368B

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