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OUR SUPPLIERS
KOMABIOTECH
301,
Gayang Technotown, #1487
Gayang 3 dong,
Gangseo-gu
Seoul 157-793, KOREA
Spherotech, Inc.
27845 Irma Lee Circle, Unit 101
Lake Forest, IL 60045
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Exalpha
Biologicals,
Inc.
2 Shaker Road,
Unit B101
Shirley, MA
01464
SCETI K.K
BIOSCIENCE Export DF Kasumigaseki Place,3-6-7 Kasumigaseki Chiyoda-ku, Tokyo 100-0013 JAPAN
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EY
Laboratories, Inc. Headquarters
107 N.
Amphlett Blvd
San Mateo, CA. 94401 USA
EXBIO Praha, a.s.
Nad
Safinou II 366
252 42 Vestec
Czech Republic
Sacace Biotechnologies S.r.l.
Via Scalabrini,
44
22100 Como Italy
GENTAUR BVBA
VAT BE0473327336
Av. de l Armee 68 B4
1040 Brussels
BELGIUM
Tel + 32 16 58 90
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GENTAUR France SARL
SIRET 48423788800017
Rue Lagrange, 9
75005 Paris,
France
Tel 01 43 25 01 50
Fax 01 43 25 01 60
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Marienbongard 20
52074 Aachen,
Germany
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Pol Sp. Z.o.o. Ulica
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Tel 00 48 58 760 77
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Fax: 00 32 16 50 90
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23015 Milano, Italy
Tel 02 36 00 65
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Other Countries
0032 (0)16 41 44 07 |
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EnzyChromTM Acetylcholine Assay Kit (EACL-100)
Quantitative Colorimetric/Fluorimetric Acetylcholine
Determination
DESCRIPTION
ACETYLCHOLINE is a neurotransmitter produced in
acetylcholinergic neurons. It plays important roles in skeletal muscle
movement, regulation of smooth and cardiac muscles, as well as in
learning, memory and mood. BioAssay Systems' method provides a simple,
direct and highthroughput assay for measuring acetylcholine in
biological samples. In this assay, acetylcholine is hydrolyzed by
acetylcholinesterase to choline which is oxidized by choline oxidase to
betaine and H2O2. The resulting H2O2 reacts with a specific dye to form
a pink colored product. The color intensity at 570nm or fluorescence
intensity (530/585 nm) is directly proportional to the acetylcholine
concentration in the sample.
KEY FEATURES
Use 20 μL samples. Linear detection range:
colorimetric assay 10 to 200 μM, fluorimetric assay 0.4 to 10 μM
acetylcholine.
APPLICATIONS
Assays: acetylcholine
in biological samples such
as serum, plasma, urine, saliva, milk, tissue, and cell culture.
Drug Discovery/Pharmacology: effects of drugs on
acetylcholine metabolism.
KIT CONTENTS
Assay Buffer:
10 mL ACHE Enzyme:
120 μL
Enzyme Mix: 120 μL Dye Reagent: 120 μL
Standard: 400 μL 2 mM acetylcholine
Storage conditions. The kit is shipped on ice.
Store all components at -20°C. Shelf life of three months after receipt.
Precautions: reagents are for research use only.
Normal precautions for laboratory reagents should be exercised while
using the reagents. Please refer to Material Safety Data Sheet for
detailed information.
COLORIMETRIC ASSAY
Sample treatment: liquid samples such as serum
and plasma can be assayed directly. Tissue and cell lysates can be
prepared by homogenization in cold 1 x PBS and centrifugation (5 min at
14,000 rpm). Use clear supernatants for assay. Milk samples should be
cleared by mixing 600 μL milk with 100 μL 6 N HCl. Centrifuge 5 min at
14,000 rpm. Transfer 300 μL supernatant into a clean tube and neutralize
with 50 μL 6 N NaOH. The neutralized supernatant is ready for assay
(dilution factor n = 1.36).
Note: (1). SH-containing reagents (e.g. b–mercaptoethanol,
dithiothreitol,> 5 μ M)
are known to interfere in this assay and should be avoided in sample
preparation. (2). This assay is based on an enzyme-catalyzed kinetic
reaction. Addition of Working Reagent should be quick and mixing should
be brief but thorough.
1. Equilibrate all components to room temperature.
Briefly centrifuge the tubes before opening. Keep thawed tubes on ice
during assay.
2.
Standards: mix 24 μL 2 mM Standard with 216 μL dH2O (final 200 μM).
Dilute standard in dH2O as follows. No 200 μM STD + H2O Vol (μL)
Acetylcholine (μM) 1 100 μL + 0 μL 100 200 2 60 μL + 40 μL 100 120 3 30
μL + 70 μL 100 60 4 0 μL +100 μL 100 0
Transfer 20 μL diluted standards into separate wells
of a clear flatbottom 96-well plate.
Samples: transfer 20 μL of each sample into
separate wells of the plate.
Note: if a sample is known to contain choline,
prepare an extra sample blank well with 20 μL
of the sample.
3. Color reaction. Prepare enough Working
Reagent by mixing, for each well, 85 μL Assay Buffer, 1 μL ACHE Enzyme,
1 μL Enzyme Mix and 1 μL Dye Reagent. Add 80 μL Working Reagent to each
well.
Note: for samples that contain choline, prepare a
blank control reagent with no ACHE Enzyme (i.e., 85 μ L
Assay Buffer, 1
μL Enzyme Mix and
1 μL
Dye Reagent). Add 80
μL
of the control Reagent to each Sample Blank well.
Immediately tap plate to mix. Incubate 20 min at room temperature.
4. Read optical density at 570nm (550-585nm).
FLUORIMETRIC ASSAY
The fluorimetric assay procedure is similar to the
colorimetric procedure except that (1) 0, 3, 6 and 10 μM acetylcholine
standards and (2) a black 96-well plate are used. Read fluorescence
intensity at lex = 530 nm and l em = 585 nm.
Note: if the calculated acetylcholine
concentration of a sample is higher than 200 μM in the Colorimetric
Assay or 10 μM in the Fluorimetric Assay, dilute sample in water and
repeat the assay. Multiply result by the dilution factor n.
CALCULATION
Subtract blank value (#4) from the standard values
and plot the DOD or DF against standard concentrations. Determine the
slope and calculate the acetylcholine concentration of Sample,
RSAMPLE and RBLANK are optical density
or fluorescence intensity readings of the Sample and H2O Blank (or
Sample Blank if sample contains choline), respectively. n is the
sample dilution factor. Conversions: 1 mM acetylcholine equals
14.6 mg/dL, 0.015% or 146 ppm.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipetting devices, centrifuge tubes, clear
flat-bottom uncoated 96-well plates, optical density plate reader; black
flat-bottom uncoated 96-well plates, fluorescence plate reader.
LITERATURE
1. Vizi, E.S. et al (1985). A simple and sensitive
method of acetylcholine identification and assay. Bioassay combined with
minicolumn gel filtration or high-performance liquid chromatography. J
Pharmacol Methods. 13:201-211.
2. Gilberstadt, M.L. and Russell, J.A. (1984).
Determination of picomole quantities of acetylcholine and acetylcholine
in physiologic salt solutions. Anal Biochem. 138:78-85.
3. Israel, M. and Lesbats, B. (1982). Application to
mammalian tissues of the chemiluminescent method for detecting
acetylcholine. J Neurochem. 39:248-250.
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QuantiChromTM Acetylcholinesterase Assay Kit
(DACE-100)
Rapid Colorimetric Determination of
Acetylcholinesterase Activity
DESCRIPTION
ACETYLCHOLINESTERASE (EC 3.1.1.7, AChE),
also known as RBC cholinesterase, is found primarily in the
blood and neural synapses. Low serum cholinesterase activity may
relate to exposure to insecticides or to one of a number of
variant genotypes. AChE catalyzes the hydrolysis of the
neurotransmitter acetylcholine into choline and acetic acid, a
reaction necessary to allow a cholinergic neuron to return to
its resting state after activation. Cholinesterase levels of
cells and plasma are used as a guide in establishing safety
precautions relative to exposure and contact, as well as a guide
in determining the need for workers to be removed from areas of
contact with the organic phosphate insecticides. Simple, direct
and automation-ready procedures for measuring AChE activity are
very desirable. BioAssay Systems' QuantiChromTM
Acetylcholinesterase Assay is based on an improved Ellman
method, in which thiocholine produced by the action of
acetylcholinesterase forms a yellow color with
5,5’-dithiobis(2-nitrobenzoic acid). The intensity of the
product color, measured at 412 nm, is proportionate to the
enzyme activity in the sample.
APPLICATIONS
Direct assays of acetylcholinesterase
activity in blood, serum, plasma, and other biological samples.
Evaluation of acetylcholinesterase inhibitors.
KEY FEATURES
Sensitive and accurate. Detection range
10 to 600 U/L AChE activity in 96-well plate assay.
Convenient. The procedure involves adding
a single working reagent, and reading the optical density at 2
min and 10 min at room temperature.
High-throughput. Can be readily automated
as a high-throughput 96- well plate assay for thousands of
samples per day.
KIT CONTENTS (100 tests in 96-well
plates)
Assay Buffer (pH 7.5): 30 mL Reagent: 240 mg
Calibrator: 4 mL (equivalent to 200 U/L)
Storage conditions. Store all reagents at
room temperature. Shelf life of at least 6 months (see expiry
dates on labels).
Precautions: reagents are for research
use only. Normal precautions for laboratory reagents should be
exercised while using the reagents. Please refer to Material
Safety Data Sheet for detailed information.
PROCEDURES
Sample preparation. Blood samples should
be diluted 40-fold in the Assay Buffer, e.g. accurately pipet 5
μ L
blood and mix thoroughly with 195 μL
Assay Buffer. Tissue or cell lysates are prepared by brief
sonication or homogenization in 0.1M phosphate buffer (pH 7.5),
followed by centrifugation at 14,000 rpm for 5 min. Use
supernatant for assay. Ideally samples should be assayed fresh.
If this is not possible, refrigerate samples and assay them
within 24 hours.
Reagent
preparation: the Working Reagent should be prepared
freshly and used within 30 min. Each reaction well requires 2 mg
reagent. Calculate the amount of reagent needed and weigh this
amount (mg) in a centrifuge tube. Add 200 μL
Assay Buffer per 2 mg reagent. Vortex to dissolve.
1.
Calibrator: transfer 200 μL
water and 200 μL
calibrator separately into wells of a clear bottom 96-well
plate.
Samples:
add 10 μL
sample per well in separate wells.
2. Reaction:
transfer 190 μL
freshly prepared Working Reagent to all sample wells and tap
plate briefly to mix. Read OD412nm
at 2 min and at 10 min in a plate reader.
3. Calculation: acetylcholinesterase activity
is calculated as follows,
Where OD10 and OD2 are the OD412nm values of
the sample at 10 min and 2 min, respectively. ODCAL and ODH2O
are the OD412nm values of the Calibrator and water at 10 min.
n is the dilution factor (n = 40 for whole blood).
The number “200” is the equivalent activity of the calibrator
under the assay conditions.
Note: if the calculated AChE
activity is higher than 600 U/L, dilute sample in Assay Buffer
and repeat this assay. Multiply the results by the dilution
factor.
Unit definition: one unit
of enzyme catalyzes the production of 1 μmole of thiocholine per
minute under the assay conditions (pH 7.5 and room temperature).
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipeting (multi-channel) devices. Clear-bottom
96-well plates (e.g. Corning Costar) and plate reader.
GENERAL CONSIDERATIONS
1. This assay is based on an enzyme-catalyzed
kinetic reaction. Addition of Working Reagent should be quick
and mixing should be brief but thorough. Use of a multi-channel
pipettor is recommended.
2. For assays in standard 1 mL cuvet, use 1 mL
water and 1 mL Calibrator, 50 μL sample + 950 μL Working
Reagent.
EXAMPLES
Two human blood samples were assayed in
duplicate using the 96- well plate protocol. The AChE activities
were 3,402 ± 163 and 3,660 ± 151 U/L.
LITERATURE
1. Magnottl, RA. et al. (1987). Measurement of
Acetylcholinesterase in Erythrocytes in the Field. Clin. Chem.
33/10, 1731-1 735.
2. Kovarik, Z et al. (2003).
Acetylcholinesterase active centre and gorge conformations
analysed by combinatorial mutations and enantiomeric
phosphonates. Biochem. J. (2003) 373, 33–40.
3. Ordentlich, A. et al. (1996). The
Architecture of Human Acetylcholinesterase Active Center Probed
by Interactions with Selected Organophosphate Inhibitors. J.
Biol. Chem. 271 (20): 11953–11962. |
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EnzyChromTM Choline Assay Kit (ECHO-100)
Quantitative Colorimetric/Fluorometric Choline
Determination
DESCRIPTION
CHOLINE and its metabolites play
important roles in membrane structure integrity, cellular
signaling and cholinergic neurotransmission. Aberrant regulation
in choline metabolism has been associated with mental illness
such as anxiety. BioAssay Systems' method provides a simple,
direct and high-throughput assay for measuring choline in
biological samples. In this assay, free choline is oxidized by
choline oxidase to betaine and H2O2 which reacts with a specific
dye to form a pink colored product. The color intensity at 570nm
or fluorescence intensity (530/585 nm) is directly proportional
to the choline concentration in the sample.
KEY FEATURES
Use 20 μ L
samples. Linear detection range: colorimetric assay 1 to 100
μM,
fluorimetric assay 0.2 to 10 μM
choline.
APPLICATIONS
Assays: choline in biological samples such as
serum, plasma, urine, saliva, milk, tissue, and cell culture.
Drug Discovery/Pharmacology: effects of drugs
on choline metabolism.
KIT CONTENTS
Assay Buffer: 10 mL Enzyme Mix: 120 μL
Dye Reagent: 120 μ L
Standard: 400 μL
2 mM Choline
Storage conditions. The kit is shipped on
ice. Store all components at -20°C. Shelf life of three months
after receipt.
Precautions: reagents are for research
use only. Normal precautions for laboratory reagents should be
exercised while using the reagents. Please refer to Material
Safety Data Sheet for detailed information.
COLORIMETRIC ASSAY
Sample treatment: liquid samples such as
serum and plasma can be assayed directly. Tissue and cell
lysates can be prepared by homogenization in cold 1 x PBS and
centrifugation (5 min at 14,000 rpm). Use clear supernatants for
assay. Milk samples should be cleared by mixing 600 μ L
milk with 100 μL
6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300
μL
supernatant into a clean tube and neutralize with 50
μL
6 N NaOH. The neutralized supernatant is ready for assay
(dilution factor n = 1.36).
Note: (1). SH-containing
reagents (e.g. b–mercaptoethanol, dithiothreitol, > 5
μ M)
are known to interfere in this assay and should be avoided in
sample preparation.
(2). This assay is
based on an enzyme-catalyzed kinetic reaction. Addition of
Working Reagent should be quick and mixing should be brief but
thorough.
1. Equilibrate all components to room
temperature. Briefly centrifuge the tubes before opening. Keep
thawed tubes on ice during assay.
2. Standards: mix 12 μ L
2 mM Standard with 228 μL
dH2O (final 100 μM).
Dilute standard in dH2O as follows.
Transfer 20 μ L
diluted standards into separate wells of a clear flatbottom
96-well plate.
Samples: transfer 20 μ L
of each sample into separate wells of the plate.
3. Color reaction.
Prepare enough Working Reagent by mixing, for each reaction
well, 85 μL
Assay Buffer, 1 μL
Enzyme Mix and 1 μL
Dye Reagent. Add 80 μL
Working Reagent to each well. Tap plate to mix. Incubate 20 min
at room temperature.
4. Read optical density at 570nm (550-585nm).
FLUORIMETRIC ASSAY
The fluorimetric assay is 10 times more
sensitive than the colorimetric method. The procedure is similar
to that for the Colorimetric Assay except that (1) 0, 3, 6 and
10 μ M
choline standards and (2) a black 96- well plate are used. Read
fluorescence intensity at lex
= 530 nm and l em = 585 nm.
Note: if the calculated
choline concentration of a sample is higher than 100 μ M
in the Colorimetric Assay or 10 μM
in the Fluorimetric Assay, dilute sample in water and repeat the
assay. Multiply result by the dilution factor
n.
CALCULATION
Subtract blank value (#4) from the standard
values and plot the DOD or DF against standard concentrations.
Determine the slope and calculate the choline concentration of
Sample,
RSAMPLE and RBLANK are optical
density or fluorescence intensity readings of the Sample and H2O
Blank, respectively. n is the sample dilution factor.
Conversions: 1 mM choline equals 10.4 mg/dL,
0.010% or 104 ppm.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipetting devices, centrifuge tubes, clear
flat-bottom uncoated 96-well plates, optical density plate
reader; black flat-bottom uncoated 96-well plates, fluorescence
plate reader.
LITERATURE
1. Lartillot, S. (1987). A simplified method of
production of choline oxidase suitable for choline assay. Prep
Biochem. 17:283-295.
2. Gilberstadt, M.L. and Russell, J.A. (1984).
Determination of picomole quantities of acetylcholine and
choline in physiologic salt solutions. Anal Biochem. 138:78-85.
3. Zeisel, S.H. and Millington, W.R. (1978).
Free and choline assay. Am J Clin Nutr. 31:1978-1981. |
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