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EnzyChromTM Glutamate Assay Kit
(EGLT-100)
Quantitative Colorimetric
Determination of Glutamate at 565 nm
DESCRIPTION
Glutamate is an important chemical in
general metabolism. It is also a crucial mammalian
neurotransmitter that is believed to be involved in a
number of neurological and psychiatric disorders such as
lateral sclerosis, lathyrism, autism and Alzheimer’s
disease. Glutamate is also widely used as a flavor
enhancer in the food industry. Simple, direct and
automation-ready procedures for measuring glutamate
concentration are very desirable. BioAssay Systems'
EnzyChromTM glutamate assay kit is based on glutamate
dehydrogenase catalyzed oxidation of glutamate, in which
the formed NADH reduces a formazan (MTT) Reagent. The
intensity of the product color, measured at 565 nm, is
proportionate to the glutamate concentration in the
sample.
APPLICATIONS
Direct Assays:
glutamate
in serum, plasma, tissue extracts and food
extract samples.
Drug Discovery/Pharmacology:
effects of drugs on glutamate levels.
KEY FEATURES
Sensitive and accurate .
Detection limit of 50 μM, linearity up to 2.5
mM
glutamate in 96-well plate assay.
Convenient. The procedure
involves adding a single working reagent, and reading
the optical density at time zero and at 30 min at room
temperature. No 37°C heater is needed.
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: 10 mL NAD Solution: 1
mL
Enzyme Mix: 120
μL
MTT Solution: 1.5 mL
Standard: 1 mL 100 mM Glutamate
Storage conditions .
Store all reagents at -20°C. 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
1. Calibration Curve. Prepare
600 μL 2.5 mM Glutamate Premix by
mixing 15
μL 100 mM
Standard and 585 μL distilled water. Dilute
standard as follows. Transfer 20
μL
standards into wells of a clear
bottom 96-well plate.
Samples: add 20 μL sample per
well in separate wells.
IMPORTANT: Serum and tissue
extract samples require a sample blank.
2. Reagent Preparation. Spin
the Enzyme Mix tube briefly before pipetting. For each
well of reaction, prepare Working Reagent by mixing 60
μL Assay Buffer, 1 μL Enzyme Mix, 5 μL NAD and 14 μL
MTT. Fresh reconstitution is recommended. Where a
sample blank in required, prepare a Blank Working
Reagent by mixing 60 μ L
Assay Buffer, 5
μL
NAD and 14
μL
MTT
(i.e. No
Enzyme Mix).
3. Reaction. Add 80 μL Working
Reagent (or Blank Working Reagent where appropriate) per
reaction well quickly. Tap plate to mix briefly and
thoroughly.
4. Read optical density (OD0) for
time “zero” at 565 nm (520-600nm) and OD30 after a
30-min incubation at room temperature.
5.
Calculation. Subtract OD0 from OD30 for the standard
and sample wells. Next, subtract the DODwater (Std 8)
from each DODstandard and DODsample to obtain the DDODs.
(Where a sample blank was required, subtract the
DODblank
from
DODsample
to obtain the
DDODsample.)
Plot the DDODstandard’s and use this standard curve to
convert the DDODsample values to sample glutamate
concentration.
Note: If the sample DDOD values are
higher than the DDOD value for the 2.5 mM glutamate
standard, dilute sample in distilled water and repeat
this assay. Multiply the results by the dilution factor.
Conversions: 1 mM glutamate =
14.5 mg/dL.
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 multi-channel pipettor is recommended.
2. The following substances interfere
and should be avoided in sample preparation: EDTA (>0.5
mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40
(>1%) and Tween-20 (>1%).
LITERATURE
1. Perez-de la Mora, M, et al (1989).
A Glutamate Dehydrogenase- Based Method for the Assay of
L-Glutamic Acid: Formation of Pyridine Nucleotide
Fluorescent Derivitives. Anal. Biochem. 180: 248-252.
2. Matsumura, H. and Miyachi S
(1980). Cycling assay for nicotinamide adenine
dinucleotides. Methods Enzymol. 69: 465- 470.
3. Graham, LT and Aprison, MH (1966).
Fluorometric determination of aspartate, glutamate, and
gamma-aminobutyrate in nerve tissue using enzymic
methods. Anal. Biochem. 15: 487-497. |
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