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 EnzyChrom^TM 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 mM, 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 mL            MTT Solution:  1.5 mL

   Standard:  1 mL 100 mM Glutamate

Storage conditions. Store all reagents at -20°C. Shelf life: 6 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

1. Calibration Curve. Prepare 600 mL 2.5 mM Glutamate Premix by mixing 15 mL 100 mM Standard and 585 mL distilled water. Dilute standard as follows. Transfer 20 mL standards into wells of a clear bottom 96-well plate.

    Sampl, , , es: add 20 mL 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 mL Assay Buffer, 1 mL Enzyme Mix, 5 mL NAD and 14 mL MTT. Fresh reconstitution is recommended. Where a sample blank in required, prepare a Blank Working Reagent by mixing 60 mL Assay Buffer, 5 mL NAD and 14 mL MTT (i.e. No Enzyme Mix).

3. Reaction. Add 80 mL 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 DOD_water (Std 8) from each DOD_standard and DOD_sample to obtain the DDODs. (Where a sample blank was required, subtract the DOD_blank from DOD_sample to obtain the DDOD_sample.) Plot the DDOD_standard’s and use this standard curve to convert the DDOD_sample 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.