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EnzyChromTM Sucrose Assay Kit
(ESUC-100)
Quantitative Colorimetric
Sucrose Determination at 565nm
DESCRIPTION
SUCROSE (C12H22O11) is a
disaccharide of glucose and fructose with an
α-1,2-glycosidic linkage. It is the most common
food sweetener and the most important sugar in
plants. In mammals, sucrose is readily digested
in the stomach into glucose and fructose, which
are rapidly absorbed into the bloodstream in the
small intestine. Simple, direct and
high-throughput assays for measuring sucrose
concentrations find wide applications. BioAssay
Systems' improved assay uses invertase to digest
sucrose into fructose and glucose. The resulting
fructose is then quantified using our fructose
assay reagent. The measured color intensity at
565nm is directly proportional to the sucrose
concentration in the sample.
KEY FEATURES
No interference by glucose.
Use 20 μL samples. Linear detection range: 17 to
2000 μM sucrose.
APPLICATIONS
Assays:
sucrose in biological samples (e.g. serum,
plasma, urine, saliva, milk, culture medium),
food, juice, beverage and other agricultural
products.
Drug Discovery/Pharmacology:
effects of drugs on sucrose metabolism.
KIT CONTENTS
Assay Buffer:
10
mL Invertase: 120 μL
PMS Solution: 1.5 mL
Enzyme: 120 μL
MTT Solution: 1.5 mL
Standard: 400 μL 40 mM Sucrose
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.
ASSAY PROCEDURE
Note: (1) The following
substances interfere and should be avoided in
sample preparation: ascorbic acid, SDS (>0.2%),
sodium azide, NP-40 (>1%) and Tween-20 (>1%).
(2) This assay is based on a kinetic reaction.
To ensure identical incubation time, addition of
Working Reagent to standard and samples should
be quick and mixing should be brief but
thorough.
Sample treatment: liquid
samples such as serum, plasma and fruit juices
can be assayed directly. 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).
1. Equilibrate all components
to room temperature. Briefly centrifuge the
tubes before opening. Keep thawed tubes on ice
during assay.
2. Standards: mix 12 μL
40 mM Standard with 228 μL dH2O (final 2000 μM).
Dilute standard in dH2O as follows.
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 fructose, prepare an extra sample blank
well with 20 μ L
of the sample.
3. Color reaction.
Prepare enough Working Reagent by mixing, for
each reaction well, 56 μL Assay Buffer, 1 μL
Invertase, 1 μL Enzyme, 14 μL PMS Solution and
14 μL MTT Solution. Add 80 μL
Working Reagent to each well.
Note: for the sample that
contains fructose, prepare a blank control
reagent with no Invertase (i.e., 56 μ L
Assay Buffer, 1
μL
Enzyme,
14
μL
PMS Solution and 14
μL
MTT Solution). Add 80
μL
of the
control Reagent to each Sample Blank well.
Immediately tap plate to mix.
Incubate 60 min at room temperature.
4. Read optical density at
565nm (520-600nm).
Note: If the calculated
sucrose concentration of a sample is higher than
2000 μM, dilute sample in water and repeat the
assay. Multiply
result by the dilution factor
n.
CALCULATION
Subtract blank value (water,
#4) from the standard values and plot the DOD
against standard concentrations. Determine the
slope and calculate the sucrose concentration of
Sample,
ODSAMPLE, OD BLANK
are
optical density values of the sample and H2O
Blank (or Sample Blank if sample contains
fructose), respectively. n is the sample
dilution factor.
Conversions: 1 mM sucrose
equals 34.2 mg/dL, 0.034% or 342 ppm.
MATERIALS REQUIRED, BUT NOT
PROVIDED
Pipetting devices, centrifuge
tubes, clear flat-bottom uncoated 96-well
plates, optical density plate reader.
LITERATURE
1. Harrison, C.S. and Faler,
K.T. (1957). Analysis of the chemical sucrose
determination of extracellular fluid volume
using C14-labeled sucrose. Am. J. Physiol.
188:568-570.
2. Holmes, E.W. (1997).
Coupled enzymatic assay for the determination of
sucrose. Anal. Biochem. 244: 103-109.
3. Young, M.K. and Prudden,
J.F. (1954). Simultaneous determination of
sucrose and inulin in biologic fluids. J. Lab.
Clin. Med. 44:160-165. |
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