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. 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 50uM, linearity up to 2.5mM 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: 10mlNAD Solution:1ml Enzyme Mix: 120ulMTT Solution: 1.5ml Standard: 1ml 100mM GlutamateStorage 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 600ul 2.5mM Glutamate Premix by mixing 15ul 100mM Standard and 585ul distilled water. Dilute standard as follows. Transfer 20ul standards into wells of a clear bottom 96-well plate
No Premix+H2O Vol (ul) Glutamate (mM)1 100ul+0ul 100 2.52 80ul+20ul 100 2.03 60ul+40ul 100 1.54 40ul+60ul 100 1.05 30ul+70ul 100 0.756 20ul+80ul 100 0.57 10ul+90ul 100 0.258 0ul+100ul 100 0Samples: add 20ul 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 60ul Assay Buffer, 1ul Enzyme Mix, 5ul NAD and 14ul MTT. Fresh reconstitution is recommended. Where a sample blank in required, prepare a Blank Working Reagent by mixing 60ul Assay Buffer, 5ul NAD and 14ul MTT (i.e. No Enzyme Mix)
3. Reaction. Add 80ul 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
[Glutamate] =DDODSAMPLESlope (mM)Note: If the sample DDOD values are higher than the DDOD value for the 2.5mM glutamate standard, dilute sample in distilled water and repeat this assay. Multiply the results by the dilution factor
Conversions: 1mM 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 (>0.5mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).
