Glutamine Assay Kit, BioAssay(TM)

Glutamine is an amino acid synthesized in the muscle that plays major roles in protein synthesis, acid-base balance, anabolic processes and is utilized for cellular energy and as a carbon source. It is used in treatment of injury, trauma, burns, and also as a supplement for muscle growth and post-surgery healing.\n\nSimple, direct and automation-ready procedures for measuring glutamine concentration are very desirable. Glutamine assay kit is based on hydrolysis of glutamine to glutamate and colorimetric determination of the product. The intensity of the product color, measured at 565 nm, is proportional to the glutamine concentration in the sample.\n\nApplications:\nDirect Assays: glutamine in serum, plasma, urine, tissue extracts and cell culture samples.\nDrug Discovery/Pharmacology: effects of drugs on glutamine metabolism.\n\nKey Features:\nSensitive and accurate. Use 20ul sample. Linear detection range 0.023-2mM glutamine in 96-well plate assay.\nConvenient. The procedure involves adding a single working reagent, incubation for 40 min at room temperature, adding a stop reagent and reading the optical density. No 37 degrees C heater is needed.\nHigh-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.\n\nKit Contents: (100 tests in 96-well plates)\nAssay Buffer: 15ml NAD Solution: 1ml\nEnzyme A: 120ul MTT Solution: 2 x 1.5ml\nEnzyme B: 220ul Stop Reagent: 25ml\nStandard: 400ul 100mM Glutamine\nStorage conditions. Store all reagents at -20 degrees C. Shelf life of 3 months after receipt.\nPrecautions: 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.\n\nProcedure:\nNote: (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: ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).\n1. Standard Curve. Prepare 2.0mM glutamine Premix by mixing 5ul 100mM Standard and 245ul distilled water. Dilute standard as follows. Transfer 20ul standards into wells of a clear flat-bottom 96-well plate.\nNo Premix+H2O Vol (ul) Glutamine (mM)\n1 100ul+0ul 100 2.0\n2 60ul+40ul 100 1.2\n3 30ul+70ul 100 0.6\n4 0ul+100ul 100 0.0\nSamples: add 20ul sample per well in separate wells.\nImportant: if a sample is known to contain glutamate, a sample blank control is required. In this case, transfer an additional 20ul sample into a separate well.\n2. Reaction. Spin the enzyme and reagent tubes briefly before pipetting. Fresh reconstitution is recommended. For each standard and sample well, prepare Working Reagent by mixing 65ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B, 2.5ul NAD and 14ul MTT. Where a sample blank is required, prepare a Blank Working Reagent by mixing 65ul Assay Buffer, 1ul Enzyme B, 2.5ul NAD and 14ul MTT (i.e. No Enzyme A). Add 80ul Working Reagent per well to standards and sample wells. Where appropriate, add 80ul Blank Working Reagent to the Sample Blank wells. Tap plate to mix briefly and thoroughly.\n3. Incubate 40 min at room temperature. Add 100ul Stop Reagent to each well. Read OD at 565 nm (520-600 nm).\n\nCalculation:\nSubtract water (#4) blank OD from OD values for the standards. Plot DOD against standard concentrations. Determine the slope and calculate sample glutamine concentration,\n[Glutamine] =\nODSAMPLE

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SPECIFICATIONS

Catalog Number

G7120-05

Size

1Kit

References

1. Cattaneo, MV and Luong, JH. (1993). Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor. Biotechnol Bioeng. 41(6): 659-665.\n2. Messer, M. (1955). A simple method for the estimation of glutamine in brain extracts. Biochim Biophys Acta. 17(1): 151-152.\n3. Foss, OP. (1952). A new growth medium for the cultivation and production of Clostridium welchii SR 12 in Krebs' method for the quantitative determination of glutamine and glutamic acid. Scand J Clin Lab Invest. 4(4): 371-372.