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Featured Customer: Sheena D'Arcy

Structural biology and SEC-MALS, go together like… horses and stalls? One might be tempted to make the analogy and rather unfortunate rhyme, especially in horse country. The University of Texas, Dallas is where Prof. Sheena D’Arcy has set her stake in the ground, and SEC-MALS is as essential to her work on protein structure, dynamics and function as, well, a horse is to a cowboy.

Prof. D’Arcy is interested in high-resolution studies of large protein complexes involved in transcription initiation and chromatin dynamics. These complexes are master regulators of gene expression and dictate cell identity, so understanding how they work at the molecular level will have broad repercussions for regenerative medicine, as well as for diseases such as cancer that are associated with aberrant gene transcription.

After expressing the proteins recombinantly, the D’Arcy lab performs a variety of biochemical, biophysical, and structural studies to:

  • measure binding affinity and stoichiometry of the complexes
  • determine the dynamics of the proteins in solution
  • identify of the key interfaces
  • see molecular structure

Multiple experimental techniques are applied, including HDX-MS and x-ray crystallography. As might be expected, light scattering supports this research extensively. The miniDAWN TREOS and Optilab T-rEX were some of the first pieces of equipment ordered when the D’Arcy lab was established in August 2015, for use downstream of a size-exclusion chromatography column. The SEC-MALS setup is used at several stages in the research pipeline and is almost always in use;

  1. To screen additives and buffer conditions for challenging proteins that aggregate during purification, and to determine overall molecular weights and polydispersity;
  2. To determine the oligomeric state of single proteins. Many of the proteins and oligomers are non-globular and elongated, so simply using analytical SEC, which separates by hydrodynamic volume, would give misleading results for molar mass. Some of the proteins also form homo-oligomers in a salt-dependent manner, and SEC-MALS provides for characterization across a range of protein and salt concentrations.
  3. To determine the absolute stoichiometry of complexes, by performing titrations of each binding partner and quantifying the relative amounts of free and bound species identified based on their molecular weights and elution volumes. Prof. D’Arcy notes that they have done the latter for an almost 300 kDa complex containing 10 protein chains! A quick SEC-MALS analysis of a mutant is also useful for determining if the mutations influence complex integrity.

Sheena DArcy

   

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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