Katherine Bowers

Wyatt CG-MALS is a core technique that is invaluable for a protein scientist!

The Calypso II instrument, used in combination with a Wyatt Multi-Angle Light Scattering (MALS) instrument and a Differential Refractive Index instrument, allow for the collection of Composition-Gradient MALS data (CG-MALS). The instrument works as stopped-flow mixer to introduce protein samples, as a function of concentration, into the detectors. Examination of the changes in light scattering and the absolute MW can provide invaluable insight into molecular associations (protein-protein or protein-other molecule). In our laboratory, our main area of investigation is the formation of higher order structures as a function of protein concentration, information that is paramount in the development of both purification processes and formulation selection. The self-interaction application allows for the measurement of the interaction parameter (A2 or B22). The user is able to analyze the data by several different association models to determine how the molecules are interacting and how the size and population of the oligomers change with protein concentration and/or buffer conditions. The instrument is user-friendly, very robust and easy to maintain. The most valuable thing that I have gained from the instrument, aside from high quality results, is how much I have learned from the Wyatt application scientists regarding the fundamentals of protein interactions and how to interpret CG-MALS data. This knowledge goes beyond the use of the Calypso II instrument into other characterization methods that we use and the way we set up experiments and interpret results. Wyatt Technology is a great company to work with!

Application Area: Analysis of protein-protein interactions and protein self-association

D. Brent Halling

Phenomenal service from hard working technical support staff

Binding studies with the Calypso are easy to set up, take only a few hours, and are easy to interpret. The technical support from Wyatt is both deeply and broadly knowledgeable. There are few places that offer the scope and quality of training Wyatt provides. I use Calypso II to measure both the stoichiometry and association constants of protein-protein interactions. Most binding methods have readouts that do not have physical meaning, such as maximum amplitude or slope of binding curve. Another major common problem is that models that describe the data contain parameters that are non-identifiable. Most experiments on the Calypso are not subject to the same issues. The determination of mass by the light scattering is not subject to the same concerns as other binding assays. 1) The mass is calculated on a calibrated instrument where all other parameters that affect light-scattering can be measured. 2) The structure that the Calypso data report has a structure that is informative on both the stoichiometry and the Kd. No more slopes or maxima! Even compared to the "Gold standard" analytical ultracentrifugation, Calypso (or composition gradient multi-angle light scattering CG-MALS) is superior. We found that non-1:1 stoichiometries of protein complexes can be very difficult to analyze with AUC data, whether using equilibrium sedimentation or velocity sedimentation. Although we get consistent results for the sedimentation rate for particles, the calculation of mass from any AUC data can run into identifiability issues. In contrast, experiments with CG-MALS can be designed to directly explore which stoichiometries can form at specific molar ratios of each particle. Furthermore, less sample and far less time is required. I strongly recommend using Wyatt instruments for particle characterization. Competitors like Malvern have not caught up and they do not provide any instrument that does anything comparable to the Calypso II. Wyatt provides unmatched service beyond the purchase of the instrument. We have used Wyatt instruments for over a decade without any drop in their excellent support.

Application Area: Protein interactions