Analysis Metabolism Molecular Modelling
(04 72 43 80 85)
Manager: Isabelle Rahioui (TR INRA, 100%)
Scientific advisors: Pedro Da Silva
Objectives: This technical platform is dedicated to the identification and quantification of amino acids in biological extracts. The extracts may be processed directly to analyze their composition in terms of free amino acids, or after hydrolysis, to determine the amino acid composition of their peptides and proteins. The equipment has also been adapted to measuring the radioactive activity of amino acids during experiments involving radioactive tracers (14C, 35S and 3H). The aim of these amino acid analyses is to respond, for example, to the need for quantification in metabolic studies or nutritional assessments in various organisms.
Techniques and equipment: Amino acids are separated by reversed-phase, high-performance liquid chromatography after pre-column derivation with orthophthalaldehyde (OPA) or with fluorenylmethyl chloroformiate (FMOC), and then detected and quantified by fluorimetry: excitation at 340 nm with detection at 450 nm for the primary amino acids derived using OPA; excitation at 266 nm with detection at 305 nm for secondary amino acids derived using FMOC. The analyses are carried out using a minimum volume of about 10 µL and, for each of the amino acids identified, the quantification limit is of the order of 20 pmol/µL.
The equipment currently available consists of an HPLC system (Agilent 1100) equipped with an automatic sampler and a fluorescence detector. An HCl vapor-phase hydrolysis station (Waters Pico-Tag) and a flow scintillation counter (Lablogic ß-RAM 4) complete this analytical system.
Access conditions: This analytical platform is available (depending on the workload) for use by groups from outside the team in various ways:
i) Internal IFR service. The analyses are carried out by the engineer responsible for the platform and the results are sent to the requesting laboratory for interpretation. The fee charged consists solely of the real cost of the analyses, the labor costs being met by UMR BF2I.
ii) Collaboration with UMR BF2I. For the joint projects carried out, one or more members of Unit BF2I may be involved in setting up the protocol and interpreting the results. The cost of the analyses (running costs and labor costs) are met by Unit BF2I, the members of the Unit who take part in the project are co authors of the resulting publications.
iii) External service provision after a contract has been signed. After analysis, the results are returned to the customer who owns them. The billing includes the real cost of the analyses plus the labor costs.
Localisation: This “Analysis Metabolism Molecular Modelling” platform is located at UMR203 BF2I, in the Louis Pasteur Building at the Lyon INSA (Pasteur, Level 1, Room 406.01.08).