Glutamine Assay Kit

Produktnummer ABIN1000310

Produktdetails

Target: GFPT2 (Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2))

Applikation: Biochemical Assay (BCA)

Proben: Serum, Plasma, Urine, Tissue Homogenate, Cell Culture Supernatant

Spezifität: 23 μM

Produktmerkmale: Sensitive and accurate. Use 20 µL sample. Linear detection range 0.023 - 2 mM glutamine in 96-well plate assay.
Convenient. 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°C heater is needed.
High-throughput. Can be readily automated as a high-throughput 96- well plate assay for thousands of samples per day.

Bestandteile: Assay Buffer: 15 mL. NAD Solution: 1 mL. Enzyme A: 120 µL. MTT Solution: 2 x 1.5 mL. Enzyme B: 220 µL. Stop Reagent: 25 mL. Standard: 400 µL 100 mM Glutamine.

Benötigtes Material: Pipeting (multi-channel) devices. Clear flat-bottom 96-well plates (e.g. Corning Costar) and plate reader.

Anwendungsinformationen

Applikationshinweise: Direct Assays: glutamine in serum, plasma, urine, tissue extracts and cell culture samples.
Drug Discovery/Pharmacology: effects of drugs on glutamine metabolism.

Protokoll: Note: (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%).
1. Standard Curve. Prepare 2.0 mM glutamine Premix by mixing 5 µL 100 mM Standard and 245 µL distilled water. Transfer 20 µL standards into wells of a clear flat-bottom 96-well plate. Samples: add 20 µL sample per well in separate wells. IMPORTANT: if a sample is known to contain glutamate, a sample blank control is required. In this case, transfer an additional 20 µL sample into a separate well.
2. 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 65 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 2.5 µL NAD and 14 µL MTT. Where a sample blank is required, prepare a Blank Working Reagent by mixing 65 µL Assay Buffer, 1 µL Enzyme B, 2.5 µL NAD and 14 µL MTT (i.e. No Enzyme A). Add 80 µL Working Reagent per well to standards and sample wells. Where appropriate, add 80 µL Blank Working Reagent to the Sample Blank wells. Tap plate to mix briefly and thoroughly.
3. Incubate 40 min at room temperature. Add 100 µL Stop Reagent to each well. Read OD at 565 nm (520-600 nm).

Ergebnisberechnung:

Subtract water (#4) blank OD from OD values for the standards. Plot OD against standard concentrations.
Conversions: 1 mM glutamine = 14.6 mg/dL or 146 ppm.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Referenzen

Behjousiar, Constantinou, Polizzi, Kontoravdi: "FIBS-enabled noninvasive metabolic profiling." in: Journal of visualized experiments : JoVE, Issue 84, pp. e51200, (2014) (PubMed).

Tang, Wang, Wang, Zhou, Zhang, Li, Chen: "Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification." in: Journal of cellular biochemistry, Vol. 113, Issue 2, pp. 518-27, (2012) (PubMed).

Behjousiar, Kontoravdi, Polizzi: "In situ monitoring of intracellular glucose and glutamine in CHO cell culture." in: PLoS ONE, Vol. 7, Issue 4, pp. e34512, (2012) (PubMed).

Lin, Chen, Kensicki, Li, Kong, Li, Mohney, Shen, Stiles, Mizushima, Lin, Ann: "Autophagy: resetting glutamine-dependent metabolism and oxygen consumption." in: Autophagy, Vol. 8, Issue 10, pp. 1477-93, (2012) (PubMed).

Antigendetails

Target: GFPT2 (Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2))

Andere Bezeichnung: Glutamine (GFPT2 Produkte)

Synonyme: gfpt1 Kit, AI480523 Kit, GFAT2 Kit, glutamine-fructose-6-phosphate transaminase 2 L homeolog Kit, glutamine-fructose-6-phosphate transaminase 2 Kit, glutamine fructose-6-phosphate transaminase 2 Kit, gfpt2.L Kit, GFPT2 Kit, gfpt2 Kit, Gfpt2 Kit

Hintergrund: Quantitative determination of glutamine by colorimetric (565nm) method.
Procedure: 40 min.

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. Simple, direct and automation-ready procedures for measuring glutamine concentration are very desirable. This 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.