Objectives

To measure the molecular weight and the hydrodynamic and thermodynamic properties of a protein or other macromolecule.

People

Dr. Soumya De

Facilitator

School of Bio Science

somde@iitkgp.ac.in

+91-3222-214552

Equipment Details

Analytical Ultracentrifuge

Utility and Working Principal

The analytical ultracentrifuge is at present the most versatile, rigorous and accurate means for determining the molecular weight and the hydrodynamic and thermodynamic properties of a protein or other macromolecule. There is no other technique that is able to provide the same range of information with this precision and accuracy. This is because the method of sedimentation analysis is based on the sound platform of thermodynamics. All terms in the equation describing sedimentation behavior are experimentally determinable.

Many macromolecules including proteins have an inherent property to form aggregates which changes the molecular weight of the species formed, and analytical centrifugation provides valuable information regarding the molecular weight of the species. Analytical centrifugation can directly measure the molecular weights of solutes without having to rely on calibration or making assumptions concerning shape. It can detect a wide range of molecular weights ranging from small proteins to larger aggregates and also larger protein present in virus. Moreover the molecular weights of calibration standards used in electrophoresis prepared using sequencing techniques, for which analytical centrifugation are the best method.

Biomacromolecular recognition including its interactions with small molecules is an important part of bioscience. The sedimentation equilibrium methodology in the analytical ultracentrifuge is the only technique presently capable of analyzing such interactions over a wide range of solute concentrations, without interference in the chemical equilibrium. Weak interactions, in the order of 10-100 M^-1 (association constant, Ka), cannot be studied using electrophoresis but can be effectively done with analytical centrifugation. Such interactions are also studied using absorbance, frequently by labeling with a chromophore. These can be easily studied using an analytical centrifuge.

Analytical ultracentrifuge is also a powerful technique for the characterization of nanomaterials, especially in the small size ranges (i.e., 1−10 nm). By centrifuging the particles at high centrifugal forces, it is possible to follow their concentration depletion profiles over time in order to determine the particle sedimentation coefficients, S. The small (1−10 nm) nanoparticles are the most difficult to detect but often the most biologically and environmentally relevant. Furthermore, the particle friction coefficient can also be determined in order to gain critical information on the particle form i.e. shape.

Detection mode -    a) Built-in scanning UV/Visible    b) Built-in Rayleigh interference optics

Sample holder - Preassembled cells for the two rotors: with sapphire window and/or quartz window