Abstract
High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed.
Highlights
There is no structural biology laboratory that can be functional without a chromatography system which is used for the purposes of purifying macromolecules [1,2,3,4]
One of the major purification methods used is size exclusion chromatography (SEC) which is based on the shape and size of the eluted macromolecules, such that during a SEC experiment larger macromolecules are eluted faster than the small ones which are retarded within the stationary hydrophilic resins [5]
Since the two proteins used in this study have well-defined molecular weights and the measured molecular masses are volume averaged, we have interpreted deviations in the measured molecular masses to represent the degree of assembly in the mobile phase under examination (i.e., the A22 value of Equation (1) or B22 value of Equation (2))
Summary
There is no structural biology laboratory that can be functional without a chromatography system which is used for the purposes of purifying macromolecules [1,2,3,4]. Common wisdom is to calibrate gel filtration columns using a standard mobile phase (such as phosphate buffered saline) and use several standard proteins of known molar masses in order to create a calibration diagram (Log molar mass vs elution time/volume) [6,7] These diagrams are used to retrieve information about macromolecules under investigation; including estimates of their molar masses, degree of oligomerization, and stability [8]. While it has long been known that solution properties can influence the elution time of a macromolecule under SEC, it remains commonplace not to account for these solution properties before consulting a calibration curve—with many laboratories performing a single calibration curve that is applied to all subsequent buffers used This can result in errors in estimated molar masses that may lead to the incorrect assignment of oligomeric state or other macromolecular solution properties [14,15]. This can be overcome through the use of static light scattering (SLS) measurements in-line with SEC experiments that can provide a direct measurement of Crystals 2017, 7, 331; doi:10.3390/cryst7110331 www.mdpi.com/journal/crystals
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