Analytical Ultracentrifugation AUC

Presentation

Analytical ultracentrifugation (AUC) studies macromolecules in solution that are subjected to a centrifugal field. AUC combines the separation of the macromolecules and the analysis of their transportation in view of a rigorous thermodynamics. AUC is particularly suitable for studying the interactions of membrane proteins, glycosylated proteins, complexes between proteins and polymer or ANR... Sedimentation velocity and sedimentation equilibrium experiments allow :

• assessing sample homogeneity
• determining the association state of complexes
• providing indications on the general shape of the macromolecules
• quantifying association constants and weak inter-molecular interactions that determine protein solubility.
• analysing complex multi-component systems, on the basis of their density and optical properties (absorbance, refractive index and fluorescence).

The AUC platform is committed to the establishment of a quality approach implementation that resulted in June 2011 with the ISO 9001 (2008) and in July 2015 with NF X50-900 (2015).

Key words

Analytical ultracentrifugation, sedimentation velocity, sedimentation equilibrium, molar mass, hydrodynamic radius, interactions, composition.

Staff

Manager : Christine EBEL / Caroline Mas
Operated by : Aline Le Roy

Equipment

• An analytical ultracentrifuge Beckman XLA/XLI with a double detection system (absorbance and interference) equipped with rotors ANTi-60 and ANTi-50 allowing the simultaneous measurement of seven samples
• A fluorescence detector (Aviv)
• A density-meter (Anton PAAR) DMA 5000
• A viscosity-meter (Anton PAAR) AMVn
• Used softwares : SEDFIT and SEDPHAT (created by P. Schuck, USA), SEDNTERP (D. Hayes, T. Laue, J. Philo, USA), GUSSI and REDATE (C.A. Brautigam, USA)
  The report will mention the necessary information on instrumentation.
  Reliability tests are :
• Density-meter : Measurement of water and air (standard) before use
• Viscosity-meter : Measurement of water (standard) before use
• AUC : Test of lamp intensity each week ; annual maintenance

Access mode

• Access to instrument (for authorized persons)
• or : Data acquisition without analysis
• or : Data acquisition with analysis
• Open to academics and private companies depending on equipment and staff disponibility.
• On a service or collaboration mode, depending on technical and scientific implication.

How to make a request ?

Fisrt step is to contact us by web site, mail or telephone. The project feasibility is discussed.
The applicant should then fill the fist part of the Request form, which will then filled by the platform and validated by the two parties.
All requests should be accompanied by a form.
Example of a filled form.

Contact et location

• Contact Aline Le Roy, Christine Ebel ou Caroline Mas
• location : IBS room 323A and 327, from Monday to Friday, 8h-17h, with assistance and scientific and technical expertises.

Samples

• For more details see : « Sample and Solvent requirements for AUC ».
• If samples are sent, or stored by the plateform, storing is as defined by the applicant in the sample file (to be filled and given with the samples).
• The sample has typically a concentration in the 0.1-10 mg/mL range, for a volume between 70 et 450µl.
• The solvent contains typically 100mM salt.
• The reference solvant should have the same volume and composition as the sample ; it should not contain detergent in the case of the study of a membrane protein. A volume of 1.5 mL is required for density and viscosity measurements.

Report-results

• The report is adapted to the applicant, to be defined by the applicant in the Request form
• Results are delivered by Email, with raw data and related files.
• Report is checked by the responsible before delivery.
• Data are archived for 5 years.

Cost and delay

• Academics : participation to maintenance fees ; private companies : quotation under request.
• The date for the report delivery is fixed with the applicant (between some weeks and months).

Follow up / Development

• In the general case users and applicants must mention the platform in their publications : « This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). We thank Aline Le Roy and/or Christine Ebel, for assistance and/or access to the Analytical Ultracentrifugation (AUC) platform ».
• The person responsible for the platform is a co-author of the papers if he participates to redaction.

List of our book chapters, protocols, and methodological publications concerning AUC, SEC-MALS and SANS

List of publications