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KOMABIOTECH
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Lake Forest, IL 60045
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Exalpha
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2 Shaker Road,
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Shirley, MA
01464
SCETI K.K
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Laboratories, Inc. Headquarters
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EnzyChromTM Triglyceride Assay
Kit (Cat# ETGA-200)
Quantitative Colorimetric
Triglyceride Determination at 570nm
DESCRIPTION
TRIGLYCERIDE, also known
as TRIACYLTRIGLYCERIDE or TRIACYLGLYCERIDE, is
the main constituent in vegetable oil and animal
fats. Triglycerides play an important role as
energy sources and transporters of dietary fat.
In the human body, high levels of triglycerides
in the bloodstream have been linked to
atherosclerosis, heart disease and pancreatitis.
Simple, direct and automation-ready procedures
for measuring triglyceride concentrations find
wide applications in research and drug
discovery. BioAssay Systems' triglyceride assay
uses a single Working Reagent that combines
triglyceride hydrolysis and glycerol
determination in one step, in which a dye
reagent is oxidized to form a colored product.
The color intensity at 570nm is directly
proportional to triglyceride concentration in
the sample.
KEY FEATURES
Sensitive and accurate .
Use as little as 10 μL samples. Linear detection
range 0.01 mmol/L to 1.0 mmol/L (0.88 mg/dL to
88.5 mg/dL) triglyceride.
Simple and convenient.
The procedure involves addition of a single
working reagent and incubation for 30 min at
room temperature, compatible for HTS assays.
Improved reagent stability.
The optimized formulation has greatly enhanced
the reagent and signal stability.
APPLICATIONS:
Direct Assays:
triglyceride in biological samples (e.g. serum
and plasma).
Drug Discovery/Pharmacology:
effects of drugs on triglyceride metabolism.
KIT CONTENTS
Assay Buffer:
24
mL ATP: 250 μL Dye Reagent: 220 μL
Enzyme Mix: 500 μL
Lipase: 1000 μL
Standard: 100 μL
(equivalent to 100 mmol/L Triglyceride)
Storage conditions. The
kit is shipped on dry ice. Store Assay Buffer at
4°C and other reagents 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.
PROCEDURES
Note: (1) SH-group
containing reagents (e.g. mercaptoethanol, DTT)
may interfere with this assay and should be
avoided in sample preparation; (2) if sample
contains glycerol, use BioAssay Systems'
EnzyChromTM Glycerol Assay Kit (Cat# EGLY-200)
to determine glycerol concentration and subtract
the glycerol value to yield triglyceride
concentration.
1. Equilibrate all components
to room temperature. Keep thawed Lipase and
Enzyme Mix in a refrigerator or on ice. Dilute
Standard in distilled water as follows. Transfer
10 μL diluted standards into wells of a clear
96-well plate. Diluted standards can be used for
future assays when stored refrigerated.
Serum and plasma samples
should be diluted 5-fold in dH2O and are assayed
directly. Cells and other solid samples can be
solubilized in 5% Triton X-100 ( see
Ref. 3). Transfer 10 μL
samples into separate
wells of the 96-well plate.
2. Prepare Working Reagent
for each well, by mixing 100
μL
Assay
Buffer, 2
μL
Enzyme Mix, 5
μL
Lipase, 1
μL
ATP and 1
μL
Dye
Reagent in a clean tube. Transfer 100
μL
Working Reagent into
standards and sample wells. Tap plate to mix.
3. Incubate 30 min at room
temperature. Read optical density at 570nm
(550-585nm).
Note :
1. if the Sample OD is higher than the Standard
OD at 1.0 mmole/L triglyceride, dilute sample in
water and repeat the assay. Multiply by the
dilution factor
n.
CALCULATION
Subtract ODH2O (water, #4)
from the standard OD values and plot the OD
against standard concentrations. Determine the
slope using linear regression fitting. The
triglyceride concentration of Sample is
calculated as
OD SAMPLE
and ODH2O are optical density values of the
sample and the water blank (# 4).
n
is the dilution factor. For example serum or
plasma samples are diluted 5-fold prior to
assay,
n = 5.
Conversions :
1 mmol/L triglyceride equals 88.5 mg/dL or 10
ppm.
MATERIALS REQUIRED, BUT NOT
PROVIDED
Pipeting devices, centrifuge
tubes, clear flat bottom 96-well plates (e.g.
Corning Costar) and plate reader.
LITERATURE
1. Nägele U et al (1984).
Reagent for the enzymatic determination of serum
total triglycerides with improved lipolytic
efficiency. J Clin Chem Clin Biochem.
22(2):165-74.
2. Bucolo, G. and David, H.
(1973). Quantitative determination of serum
triglycerides by the use of enzymes. Clin.
Chem.19(5): 476-482.
3. Zhu Y et al (2000).
Genomic interval engineering of mice identifies
a novel modulator of triglyceride production.
PNAS 97(3): 1137-1142. |
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