Alanine Assay Kit

Antigen: Alanine

Applikation: Enzymaktivitätsmessung (EAA)

Produktnummer: ABIN411783

Produktbeschreibung

Beschreibung: Alanine is the 2 nd most abundant of the 20 proteinogenic amino acids. Nonessential, being available from dietary sources, it plays a key role in the glucose-alanine cycle between tissues and liver. In muscle and other tissues that degrade amino acids, amino groups are pooled as glutamate by transamination. Glutamate then transfers the amino group to pyruvate via alanine aminotransferase, forming alanine and α-ketoglutarate. The alanine is passed into the blood and transported to the liver. A reverse of the alanine aminotransferase reaction takes place in liver. Pyruvate can be used in gluconeogenesis, to form glucose which may return to other tissues through the circulatory system. There appears to be a correlation between alanine levels and and higher blood pressure, energy intake, cholesterol levels, and body mass index. BioVision’s Alanine Assay Kit provides a sensitive detection method of alanine. In the kit, alanine is converted to pyruvate which is specifically detected leading to proportional color (λ=570nm: 0-10 nmol) or fluorescence (Ex/Em 535/587nm: 0-1 nmol) development. Serum concentration: ~24-76 µg/ml (~3-9 nmol/10 µl).

Anwendungen

Protokoll: 1. Alanine Standard Curve: Colorimetric: Dilute 10 μl of the 100mM Alanine standard with 990 μl DI H 2 O to generate 1 mM standard Alanine. Add 0, 2, 4, 6, 8, 10 μl of the diluted Alanine standard into a 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well standard. Bring the volume to 50 μl with Assay buffer. Fluorimetric: Dilute standard as for the colorimetric procedure, then take 100 µl of the 1 mM standard and add to 900 µl DI H 2 O to make 0.1mM Alanine standard. Add 0, 2, 4, 6, 8, 10 μl of the diluted Alanine standard into a 96-well plate to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well standard. Bring the volume to 50 μl with Assay buffer. 2. Sample Preparation: Tissues or cells (1×10 6 ) can be homogenized in 100 μl Assay Buffer centrifuge to remove insoluble material at 13,000 g, 10 minutes. 10-50 μl deproteinized serum samples can be directly diluted in the Assay Buffer. Bring sample wells to 50 μl/well with Assay Buffer in a 96-well plate. For unknown samples, we suggest testing several doses of your sample to make sure the readings are within the standard curve range. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 μl Reaction Mix containing: Alanine Measurement Background Control* 44 μl Assay Buffer 46 µl Assay Buffer 2 μl Alanine Converting Enzyme ---------------------- 2 μl Alanine Development Mix 2 μl Alanine Development Mix 2 μl Alanine Probe** 2 μl Alanine Probe * Use background control if high levels of pyruvate are suspected to be in the samples. ** For the fluorescent assay dilute the probe 10X to reduce background. Add 50 μl of the Reaction Mix to each well containing the Alanine Standard and test samples. Mix well. Incubate the reaction for 30 min at 37°C, protect from light. 4. Measure O.D. at 570 nm in a microplate reader or fluorescence using Ex/Em 535/587 nm. 5. Calculation: Correct background by subtracting the value derived from the 0 Alanine control from all sample readings (The background reading can be significant and must be subtracted from sample readings). Plot Alanine standard Curve, Alanine concentrations of the test samples can then be calculated: C = S a /S v nmol/μl, or mM where S a is the sample amount of unknown (in nmol) from standard curve, S v is sample volume (μl) added into the wells. L-Alanine Molecular Weight is 89.1 g/mol.

Beschränkungen: Nur für Forschungszwecke einsetzbar