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 α-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.
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 lem/ex=585/530nm is directly proportional to the alanine concentration in the sample.
Key Features:Sensitive and accurate. Linear detection range in 96-well plate: 1 to 200uM alanine for colorimetric assays and 0.4 to 20uM for fluorometric assays.
Applications:Direct Assays: alanine levels in plasma, serum, urine, tissue and culture media. Drug Discovery/Pharmacology: effects of drugs on alanine metabolism.
Kit Contents (100 Tests In 96-Well Plates):Developer: 6ml ALT Enzyme: 120ulDye Reagent: 120ul Cosubstrate: 600ulAlanine Standard: 400ulStorage conditions. The kit is shipped on ice. Store all kit components at -20 °C. Shelf life of six 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:Important:: 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)
Sample PreparationTissue or cell samples (2×106) can be homogenized in 100ul 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.)
Colorimetric Procedure1. Standards. First dilute the Alanine Standard to 200uM by mixing 5ul 100mM Standard with 495ul dH2O. Next, dilute standards in 1.5-mL centrifuge tubes as described in the Table. If assaying culture media with phenol red, dilute the Alanine Standard in culture media
No Premix+dH2O Alanine (uM)1 200ul+0ul 2002 120ul+80ul 1203 60ul+140ul 604 0ul+200ul 0Transfer 50ul 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: 50ul Developer, 1ul ALT Enzyme, 5ul Cosubstrate and 1ul Dye Reagent. Add 50ul 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
Fluorometric ProcedureFor fluorometric assays, the linear detection range is 0.4 to 20uM alanine
Dilute the Standards prepared in Colorimetric Procedure 1:10 in dH2O
Transfer 50ul standards and 50ul samples into separate wells of a black 96-well plate
Add 50ul Working Reagent (see Colorimetric Procedure). Tap plate to mix
Incubate 30 min at 37°C or 60 min at RT and read fluorescence at lex=530nm and lem=585nm.
Calculation:Subtract the blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the alanine concentration of the Sample,RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and Sample Blank, respectively. n is the sample dilution factor
Note: if the calculated alanine concentration is higher than 200uM for the colorimetric assay or higher than 20uM for the fluorometric assay, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n.
Conversions: 1 mg/dL alanine equals 112.2uM, 0.001% or 10 ppm.
Materials Required, But Not Provided:Pipetting devices, clear or black flat-bottom 96-well plates, plate reader or centrifuge tubes.
