Product Information Sheet (0794-008)

This is the html version of the file http://www.markergene.com/product_sheets/pis0856.pdf.
G o o g l e automatically generates html versions of documents as we crawl the web.
To link to or bookmark this page, use the following url:

http://www.google.com/search?q=cache:CQAURqsL1qIJ:www.markergene.com/product_sheets/pis0856.pdf+GUS+assays&hl=en&ie=UTF-8

Google is not affiliated with the authors of this page nor responsible for its content.

These search terms have been highlighted:

gus

assays

Page 1

Chemiluminescent

β-Glucuronidase (GUS) Detection Kit

(Product M0856)

Product Information Sheet

(0856-006)

NOTE: The following information is given as a viable methodology for use of the

Chemiluminescent GUS Detection Kit. The user may determine their own best methods

for use dependent on the specific conditions present in their experiment.

I. OVERVIEW

Reporter genes are widely used as “markers” for analysis in gene regulation and

localization, as well as for analysis of mutation altered genes. Expression of reporter

genes can be measured by immunological assay, biochemical activity assay or by

histochemical staining of cells or tissues.

The

β-glucuronidase (GUS) enzyme from E. coli (EC 3.2.1.31) has been well

documented to provide desirable characteristics as a marker gene in transformed plants

and mammalian cells. The GUS reporter gene system has many advantages including

stable expression of E. coli GUS enzyme, no interference with normal plant metabolism,

and low intrinsic GUS activity in higher plants. The enzyme is also capable of tolerating

amino-terminal additions, making it useful for study of plant organelle transport.

Various

β-glucuronic acid substrates are available for detection of GUS expression, all of

which contain the sugar D-glucopyranosiduronic acid attached by glycosidic linkage to a

hydroxyl group of a chromogenic, fluorogenic or chemiluminescent molecule. Upon

enzymatic hydrolysis, the sugar is removed, resulting in release of the detectable

molecule, and levels of gene expression can be inferred from relative color, fluorescence

or light emission levels. 1,2 Dioxetane substrates emit visible light upon enzyme

catalysis, providing rapid results with very low background and high intensity signal. An

enhancing solution is also provided with this kit to increase light production efficiency in

solution-based assays by drawing water away from the signal production site. The

Chemiluminescent

β-Glucuronidase Detection Kit provides all the necessary reagents,

buffers, substrate, and protocols for sensitive and quantitative GUS activity assays.

II. MATERIALS

A.) GUS Extraction Buffer: 50mM NaHPO

, pH 7.0, 10mM

β-mercaptoethanol, 10mM

EDTA, 0.1% (w/v) sodium lauryl sarcosine, and 0.1% (w/v) Triton X-100.

B.) GUS Assay Buffer: 2mg/mL [(4-Methoxy)-4-(3-b-D-glucopyranoic acid-4-

chlorophenyl)] spiro [1,2-dioxetane-3, 13’-tricyclo[7.3.1.0

2,7

]tridec-2,7-ene], sodium salt

(4-Cl-DIOX-GlcU) in extraction buffer. Dilute to give a 1mg/mL working solution.

Specific conditions may require higher or lower concentrations of chemiluminescent

Page 2

substrate. Optimum dilutions should be determined based on assay conditions, as

necessary.

[(4-Methoxy)-4-(3-b-D-glucopyranoic acid-4-chlorophenyl)] spiro [1,2-dioxetane-3, 13’-

tricyclo[7.3.1.0

2,7

]tridec-2,7-ene], sodium salt

C.) Enhancing Solution

D.) Purified

β-Glucuronidase Enzyme Solution: 1 unit of enzyme per 10

µL solution,

containing 4mMol/liter phosphate buffer, pH 6.8, in dH

E.) Storage and Handling. All materials should be handled with care and stored at

°C. Reagents should be stable for at least 6 months following purchase. High

background readings for blank samples may indicate decomposition.

III. PLANT ASSAY CONDITIONS

A.) Plant Extraction: Weigh 5-50mg plant tissue, add 200-500

µL of cold GUS

Extraction Buffer, and grind with mortar and pestle until homogenized. Place sample

in microcentrifuge tubes and centrifuge for 8 minutes at 8000rcf. Use supernatant to

measure total protein concentration according to Bradford (1976) or a similar

technique. Either use the plant extract immediately or store frozen at -70

°C. Do not

store the extract at -20

°C; enzyme activity is diminished by storage at -20

°C or

higher. NOTE: For other cell types (mammalian), conditions may vary. This

procedure may be modified or replaced with a different procedure as needed. Consult

with the literature references below for information about specific lysis conditions for

your cell type.

B.) Performing the assay: To a 96-well microtiter plate, add the GUS Assay buffer,

GUS Extraction Buffer, and plant extract or blank solution (extraction buffer) as

described in the table below. Allow at least four wells for each concentration of 4-Cl-

DIOX-GlcU (two with plant extract and two with extraction buffer to serve as blanks

and correct for any nonenzymatic hydrolysis of 4-Cl-DIOX-GlcU). Modify dilutions

of 4-Cl-DIOX-GlcU and/or plant extract and conditions if necessary. For example, if

Page 3

a higher 4-Cl-DIOX-GlcU concentration is needed, use the GUS Assay Buffer

undiluted to give final [4-Cl-DIOX-GlcU] of 1.6mg/mL, 1.2mg/mL, 0.8mg/mL,

0.4mg/mL, and 0.2mg/mL, respectively. Incubate plates at 38

°C for 20-30 minutes.

Add 100

µL Enhancer Solution to each well. Measure light emission without emission

and excitation filters sets. Average the values for multiple wells of each

concentration and subtract blanks. To calculate extract activity it is necessary to

perform the above assay using only one chosen concentration of 4-Cl-DIOX-GlcU,

for example 1.6mg/mL, where enhancer is added to each of four wells (two with

extract and two serving as blanks) at 10 minute intervals and luminescence of those

wells is measured and recorded. For the zero minute test points, add extract sample

to wells containing a fixed concentration of 4-Cl-DIOX-GlcU and immediately add

enhancing solution and measure light emission. Average the values for each time

interval and subtract the blank. Prepare purified enzyme standard samples, using

same conditions (i.e. same GUS Assay Buffer concentrations) as those used for plant

extract samples. Include a minimum of 5 dilutions in order to plot a standard curve.

C.) Calculations: Plot a calibration curve of 4-Cl-DIOX (product upon enzyme

hydrolysis) standards (using purified enzyme) light intensity (RLU) vs. pmol 4-Cl-

DIOX. Plot a curve of sample/extract RLU vs. time. Calculate RLU per pmol 4-Cl-

DIOX and RLU per minute of extract/cell sample. Calculate

β-glucuronidase activity

of extract in pmol 4-Cl-DIOX per minute per

µg protein (units per µg protein)

according to equation below.

Table 1. Sample assay conditions

[4-Cl-DIOX-GlcU]

(Final)

GUS Assay Buffer

(1 mg/mL 4-Cl-

DIOX-GlcU)

GUS

Extraction Buffer

Enzyme (plant

extract/purified

enzyme) or Blank

Solution

0.8 mg/mL

µL 10

µL

0.6 mg/mL

µL 30

µL 10

µL

0.4 mg/mL

µL 50

µL 10

µL

0.2 mg/mL

µL 70

µL 10

µL

0.1 mg/mL

µL 80

µL 10

µL

Sample calculation:

Activity of extract = RLU/min x reaction volume (

µL) x 1 x 1 .

(pmol 4-Cl-DIOX RLU/pmol 4-Cl- sample volume (

µL) Vol. per test (µL) extract conc.

/min/

µg protrein) DIOX (µg protein/µL)

Page 4

IV. CELLULAR ASSAY CONDITIONS

It is recommended that measurements be made in triplicate, if possible, and that the

approximate concentration range of the chemiluminescent probe be adjusted for optimum

signal and sensitivity. Previous studies have indicated that the labeling of cells is

practically independent of the initial substrate concentration in the range of about 100pM

- 2mM. Since light emission is time dependent, a time course for the experiments should

also be generated for initial trials. The user is asked to consult with the manufacturer (or

instrument manual) of the particular instrument in use for appropriate setup conditions

needed for monitoring luminescence using a microplate assay format. Typical

microscopic analysis can be performed using a long integration time (typically 5 min.) for

photographic documentation of the light emission. Focus and aspect parameters are

performed by Nomarski (visible light) prior to integration under dark conditions.

To normalize data, each cell suspension or plate is monitored at exactly the same time

(i.e. 20 min.) after equilibration with the enhancer solution. The product-forming rate is

dependent on the concentration of incorporated probe in the cell, but with concentrations

of the probe above approximately 2 µM, enzyme kinetics are typically independent of

initial concentration. The working range of the assay will need to be determined for each

individual experiment. Adjust working concentrations accordingly. A blank prepared

with all reagents, substituting corresponding non-GUS-expressing cells should be run in

parallel if possible. Alternately, a blank prepared with all reagents except cells

(substituting water or media) can be run to determine background readings for each

experiment.

CELLULAR PROTOCOL

1.) Healthy cells expressing the

β-glucuronidase gene (GUS) or endogenous

β-

glucuronidase enzyme activity are harvested from exponential growing cultures. (Note:

unhealthy cells or those grown to confluence often exhibit high background staining due

to endogenous activity or low pH conditions).

2.) Adherent cells are plated into 12-well plates at a concentration of 1 x 10

cells/well

and grown in media (+5% FCS) under sterile, humidified conditions, 37

C, 5 % CO

atmosphere, overnight.

3.) Media is removed from the wells (except for blank controls) and the cells are washed

with sterile PBS (1 x 500uL). PBS is removed from the wells by suction pipette.

4.) 250µL of diluted GUS Assay buffer is added to each well.

5.) The plates are incubated as above for 30 min.

6.) The Assay Buffer is removed and Enhancing Solution (200µL/well) is added.

Page 5

7.) Plates are read immediately using a luminescence plate reader, in luminescence mode

with optimal dynamic range and maximum integration time (typically 1.5 to 5 sec. per

sample).

8.) Readings can also be rerun at various time intervals (5, 10, 15 min.) to obtain kinetic

data for luminescence.

9.) Equivalent multiple wells are averaged. Controls are subtracted from sample well

data.

10.) A standard curve is generated for purified enzyme assay trials (see above).

11.) Relative luminescence for sample wells is compared to standard curve data, and used

to determine GUS activity in sample wells. The relative GUS activity for different cell

lines should be determined in parallel assays, using the same conditions.

NOTE: Clear tissue culture plates (Falcon 353503 or Corning (Costar) 3513) may exhibit

some optical light transfer between wells. If possible, leaving blank wells between

sample well will reduce this “cross-over” effect, and provide better results. Please

consult the manufacturers information for the availability of white or black plates for

luminescence assay.

Chemiluminescent GUS Substrate Kinetics

500000

1000000

1500000

2000000

2500000

3000000

Time (minutes)

Lum

inescence

0.8mg/mL

0.6mg/mL

0.4mg/mL

0.2mg/mL

0.1mg/mL

0.08mg/mL

0.04mg/mL

0.02mg/mL

Figure 1. Assay performed with purified β-glucuronidase enzyme. Luminescence was measured

using a Perkin-Elmer HTS 7000 Plus UV/FL/LUM Microtiterplate reader.

Page 6

REFERENCES

• Chemiluminescent 1,2-dioxetanes.

Brij P. Giri, PCT/US99/20590

• Novel stabilized formulations for chemiluminescent assays.

Brij P Giri, PCT/US01/02779

• Novel stabilized formulations for chemiluminescent assays.

Brij P Giri, PCT/US01/02779

• Single molecule detection of Alkaline Phosphatase enzyme using enhanced

chemiluminescent from 1,2-dioxetanes and water-soluble, water-insoluble or partially

water-soluble polymers.

Brij P Giri, US Patent Application, 2002/0013250

• Deuterium-based chemiluminescent 1,2-dioxetanes.

Brij P Giri, Dinesh J Dagli, PCT application pending

• Polymeric ammonium and or phosphonium salts having added p-electrons and higher

molecular weight and enhancers for chemiluminescent systems.

Brij P. Giri, US patent pending

• Kain, S.R. & Ganguly, S., Current Protocols in Molecular Biology. (1996) p. 9.6.1.

• Jefferson, R.A., et al., Proc. Natl. Acad. Sci. USA. 86 (1986) 8447-8451.

• Jefferson, R.A., et al., EMBO J. 6 (1987) 3901-3907.

• Kosugi, S., et al., Plant Sci. 70 (1990) 130-140.

• Gallagher, S.R., et al., GUS Protocols: Using the GUS Gene as a Reporter of Gene

Expression. (1992).

• Bradford, M.M., Anal. Biochem. 72 (1976) 248-254.

Page 7

Kit Contents

Description

Quantity

Part No.

Storage

Sample preparation

GUS Extraction Buffer: 50mM NaHPO

, 25mL 0856-001 C,G

pH 7.0, 10mM

β-mercaptoethanol, 10mM

EDTA, 0.1% sodium lauryl sarcosine,

and 0.1% Triton X-100

Enhancing Solution

15mL 0856-002 C,G

GUS Assay Buffer: 2mg/mL 4-4-CL-DIOX- 4mL 0856-003

C,G

GLCU in extraction buffer

Purified

β-Glucuronidase Enzyme Solution: 2mL 0856-004 C,G

1 unit per 10µL of solution

MSDS sheets

2 0856-005 N/A

Product Information sheet

0856-006 N/A

Notes: F=store at or below -18

C; R=store at room temperature; C=store cold (4

C);

L=light sensitive; D=store desiccated; FL=flammable; G=wear protective

clothing/gloves/safety glasses when using; B=avoid breathing dust/fumes.