Analytical ultracentrifugation is an important group of powerful methods (sedimentation equilibrium and velocity) which allow determine the size, the approximate overall shape and the degree of homogeneity of proteins and other biological macromolecules in solution. These methods are specially adapted for the detection and quantitative analysis (proportion of species, stoichiometry, affinity, reversibility) of interactions leading to the formation of macromolecular complexes, including protein-protein, DNA-protein, and receptor-ligand interactions [Howlett et al. (2006) Curr. Opin. Chem. Biol. 10:430-436; Lebowitz et al. (2002) Protein Sci. 11:2067-2079; Minton (2000) Exp. Mol. Med. 32:1-5; Rivas et al. (1999) Methods 19:194-212].
- The Facility at CIB has an analytical XL-I ultracentrifuge equipped with two optical detection systems: UV - VIS variable wavelength and Raleigh interference. The latter technique enables the study of macromolecules in solutions with high concentration/absorption in the UV – VIS.
- Since its foundation in 1994, this CIB Facility has given scientific and technical support in the biophysical characterization of proteins and their interactions to a large number of research groups in the CIB, CSIC other centres, universities and other centres of domestic and foreign research. Moreover, the Facility counts with the advice and collaboration of international experts in the field. In 2009 this Facility has been granted with the ISO9001 certificate in according to the AENOR code ER-0286/2009.
This Facility has also incorporated the technique of dynamic light scattering (DLS) that provide complementary information to analytical ultracentrifugation, with two main advantages, relative to other commonly used analytical methods: low sample volume requirements (15-45 µl) and quick delivery of results (<10 min).
DLS allows determining the degree of homogeneity of macromolecular complexes in solution, to measure the translational diffusion coefficient of macromolecules and to calculate their hydrodynamic radius. This instrument (DynaPro MS/X model, Wyatt Inc.) is very well designed to study proteins and macromolecular assemblies (including polymers and liposomes), has a very easy operation (cuvette) and a Peltier temperature regulation system (4-60°C) and can be used to analyze different types of macromolecules such as proteins, polysaccharides and lipid micelles.
- We have introduced a static light scattering that can work coupled to a size exclusion chromatography system (SEC-MALS), or as a workstation for the rapid detection and real-time determination of the kinetic and equilibrium properties of biological macromolecules and complexes in solution (CG-SLS). This technique is based upon the methods of static light scattering recently developed by the group of Dr. Allen Minton, NIH [Attri & Minton (2005) Anal. Biochem. 337:103-110]. Main module is a multi-angle laser light scattering detector equipped with a flow cell (DAWN EOS model, Wyatt Inc.) that is connected to a refractive index flow detector and to a triple-syringe programmable pump. The information is acquired in minutes (important when the stability of the sample is a factor to consider). Moreover, the system allows determine the association/dissociation kinetics of a given macromolecular reaction (providing that the relaxation time of the reaction is of the order of minutes).
- Recently we have done in this Facility several studies on the oligomerization and polymerization of proteins by the combination of ultracentrifugation and light scattering techniques (Monterroso et al. (2013) Methods 59: 349–362; del Castillo et al. (2011) Biochemistry, 50: 1991–2003).
Biolayer interferometry (BLI) is a simple, optical dip-and-read assay system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and lipids in real time and small sample volumes (4.5 µL). BLI provides direct binding affinities and rates of association and dissociation between a molecule, immobilized on a fiber-optic biosensor, and the analyte in solution.Since it requires nanomole amounts of sample, it is an excellent choice for the study of proteins that are challenging to isolate. BLI also has the advantage of quantifying protein concentration from heterogeneous crude lysates, since refractive index changes in the medium do not affect shifts in the interference pattern.
- The Facility has acquired a ForteBioBLItzTM instrument and a variety of disposable biosensors that includes Ni-NTA, Aminopropylsilane, Protein A, and Amine Reactive 2nd Generation.
The operation and maintenance of this facility have a considerable cost which, at the moment, has to be provided by the users (which can request their costs in their individual grants). However, in the case of analytical ultracentrifugation experiments, we are currently charging a minimal fee to cover the instrument costs and the small spare part replacements. These fees that depend upon the group’s institution (CSIC, universities, private centers, companies) include the following:
- Experimental design.
- Data acquisition setup.
- Assembly/disassembly and cleaning of cells.
- Raw data supplied as computer readable text files.
- A brief report with information about the quality of the sample (i.e., size heterogeneity, degree of association, etc.) and a preliminary gross estimate of cell-average molar masses and sedimentation coefficients. Delivery deadlines for sedimentation velocity and sedimentation equilibrium reports will be 3 and 5 working days, respectively.
Detailed analysis and interpretation of the results and subsequent preparation of figures and text for publication are not part of the fee-based service provided by this facility, but rather fall into the category of collaborative research, the conditions of which must be negotiated in advance between the principal investigator of the user’s work group and the staff of the Facility.
For more information, contact with:
- Analytical ultracentrifugation methods at CIB (in preparation).