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Protein Conjugate and Copolymer Analysis

It is possible to determine the molar mass, size, and relative polymer fractions of a copolymer using light scattering. All that is required are two additional detectors that have differing sensitivities to the constituent polymers. Traditionally, light scattering has been used in conjunction with an RI and UV detector for this purpose.

An important class of copolymers are protein conjugates. For example, researchers often need to determine the fraction of protein in glycosylated and pegylated proteins, as well as membrane protein-detergent complexes. ASTRA has native support for protein conjugate and copolymer analysis using a light scattering detector in conjunction with a UV and RI detector.


protein conjugate analysis GUI

The interface for the protein conjugate analysis is easy to use. Once the dn/dc and UV extinction values for the protein and modifier have been entered, it is possible to view the total mass and protein fraction on a slice by slice basis. ASTRA calculates the size of the complex, mass of the complex, and masses of the constituents, displaying them with rigorous uncertainties. In addition, the concentration and calculated dn/dc values are displayed for that elution volume.


The protein conjugate analysis is a rich example to display the powerful custom graphing capabilities of ASTRA. Consider the plot at right of protein fraction vs. elution volume for a membrane protein-detergent complex. Any data that ASTRA calculates can easily be plotted in this way.

Protein Fraction

Since the protein conjugate analysis uses three detectors in series, it is absolutely essential to correct for band broadening effects to get accurate results. Consider the animation below of the relative protein and modifier fractions for a membrane protein-detergent complex. The protein fraction and mass should be constant over the eluting peak. Without the band broadening correction, it is impossible to ascertain this. With ASTRA and the correction, the true fractions are instantly revealed.

Band Broadening Effects

 

   

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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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