L-Alanine Assay Kit

Produktnummer ABIN1000288

Produktdetails

Target: L-Alanine

Applikation: Biochemical Assay (BCA)

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

Spezifität: 0.4 μM

Produktmerkmale: Sensitive and accurate. Linear detection range in 96-well plate: 1 to 200 µM alanine for colorimetric assays and 0.4 to 20 µM for fluorimetric assays.

Bestandteile: Developer: 6 mL. ALT Enzyme: 120 µL. Dye Reagent: 120 µL. Cosubstrate: 600 µL. Alanine Standard: 400 µL.

Benötigtes Material: Pipetting devices, clear or black flat-bottom 96-well plates, plate reader or centrifuge tubes.

Anwendungsinformationen

Applikationshinweise: Direct Assays: alanine levels in plasma, serum, urine, tissue and culture media.
Drug Discovery/Pharmacology: effects of drugs on alanine metabolism.

Protokoll: A. Colorimetric Procedure
1. Standards. First dilute the Alanine Standard to 200 µMby mixing 5 µL 100 mM Standard with 495 µL dH2O. Next, dilute standards in1.5-mL centrifuge tubes as described in the Table. If assaying culture media with phenol red, dilute the Alanine Standard in culture media. Transfer 50 µL of each Standard to separate wells in a 96 well plate.
2. Alanine Detection. Prepare enough working reagent (WR) for 4 standards and all samples. For each reaction combine the following: 50 µL Developer, 1 µL ALT Enzyme, 5 µL Cosubstrate and 1 µL Dye Reagent. Add 50 µL of WR to each Standard and Sample well. Mix well and incubate protected from light for 30 min at 37°C or 60 min at RT.
3. Read OD570nm.

B. Fluorimetric Procedure
For fluorimetric assays, the linear detection range is 0.4 to 20 µMalanine. Dilute the Standards prepared in Colorimetric Procedure 1:10 in dH2O. Transfer 50 µL standards and 50 µL samples into separate wells of a black 96-well plate. Add 50 µL Working Reagent (see Colorimetric Procedure). Tap plate to mix. Incubate 30 min at 37°C or 60 min at RT and read fluorescence at ex = 530nm and em = 585nm.

Aufbereitung der Reagenzien:

Equilibrate Developer to desired assay temperature. The assay requires 30 min when performed at 37°C or 60 min if performed at RT (25°C).

Aufbereitung der Proben:

Tissue or cell samples (2x10^6 ) can be homogenized in 100 µL PBS. Centrifuge at 14,000 rpm for 5 min. Use clear supernatant for assay. Serum should either be diluted at least 10-fold in dH2O or deproteinated using a 10 kDa spin filter (e.g. Microcon YM-10). If planning to measure alanine in culture media, if possible avoid media with high pyruvate concentrations (DMEM, L-15, F12, etc.).

Ergebnisberechnung:

Subtract the blank value (#4) from the standard values and plot the OD or F against standard concentrations. Note: if the calculated alanine concentration is higher than 200 µM for the colorimetric assay or higher than 20 µM for the fluorimetric assay, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n.
Conversions: 1 mg/dL alanine equals 112.2 µM, 0.001% or 10 ppm.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Antigendetails

Target: L-Alanine

Substanzklasse: Amino Acid

Hintergrund: Quantitative assay of L-Alanine by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 60 min.

Alanine, a nonessential amino acid, is utilized in the glucose-alanine cycle between tissues and the liver. In tissues that metabolize amino acids, amino groups are collected as glutamate by transamination. The amine group is then transferred by alanine transaminase (ALT) from glutamate to pyruvate to form alanine and -ketoglutarate. The alanine generated is transported to the liver where a reverse ALT reaction occurs and pyruvate is regenerated. Pyruvate is converted through gluconeogenesis to glucose which can then be recirculated to the tissues. Alanine concentration may have some correlation with high blood pressure, energy intake, cholesterol levels and body mass index. This Alanine Assay Kit provides a simple, direct and automation-ready procedure for measuring alanine concentration. Alanine is converted into pyruvate which can then be directly measured. The color intensity of the reaction product at 570nm or fluorescence intensity at gamma em/ex = 585/530nm is directly proportional to the alanine concentration in the sample.