Using Appropriate Buffer Conditions Improves Sample Preparation for Structural Analysis
News Jul 29, 2013
Wyatt Technology Corporation publishes new application note highlighting a study made by Florida State University. Authored by Tsz Kin Martin Tsui, Claudius Mundoma and Hong Li from Florida State University, the paper showcases how the Wyatt DynaPro dynamic light scattering (DLS) system can be used to characterize homogeneity and stability of a heterodimeric protein complex (HPC) under various buffer conditions. The results demonstrate that the pH of the buffer solutions influences the sample homogeneity and stability. To obtain a copy of the study please click here or visit the application note library available on www.wyatt.com.
A heterodimeric protein complex (HPC) from Pyrococcus furiosus is identified for its functional role in binding RNA. HPC has a total molecular weight of 65 kDa and has a tendency to form fibril materials in vitro as observed by electron microscopy. DLS data were acquired at 20°C using the Wyatt DynaPro NanoStar Dynamic Light Scattering (DLS) system. A total of 10 measurements were performed for each sample, and the stability and homogeneity determined from the polydispersity and autocorrelation functions using DLS provided information on the biophysical property of the HPC.
Hong Li, Department of Chemistry and Biochemistry and the Institute of Molecular Biophysics, Florida State University, said “Biological macromolecules are the focus of the Institute’s research – their form, function, interactions and mechanism of action. Challenging frontier areas include characterizations of the interactions between proteins and membranes, sugars or nucleic acids. The DynaPro NanoStar is ideally suited for studies of these complexes in addition to nanoparticles, vesicles, viruses and colloids. The results from the study demonstrate that the pH of the buffer solutions influences the sample homogeneity and stability, highlighting the conditions where polymerization of HPC is thermodynamically more favorable.”
The Institute of Molecular Biophysics has a long history as a unique interdisciplinary research unit dedicated to the application of physical and chemical principles to the investigation of biological phenomena. Dr. Claudius Mundoma is the Director of the Physical Biochemistry facility which houses a broad array of biophysical technology used for macromolecular characterization of structure of macromolecules. He comments, “a technique such as Dynamic Light scattering is very efficient, requiring less time and sample than other techniques to determine particle size distribution and aggregation states of molecules.” Applications include investigations to improve vitamin C production or to re-engineer viruses for the efficient delivery of gene therapies to those with inherited disorders.
Tsz Kin Martin Tsui, comments “The instrument is straightforward to operate and can be used for a broad range of applications requiring both accuracy and high sensitivity, including investigations into the folding of proteins, assembly into larger structures, stability and dynamics. The results of this study can direct me to improve sample preparation by using appropriate buffer conditions for biophysical studies, which is particularly important for structural analysis.”
The DynaPro NanoStar system from Wyatt Technology is one of the most widely used dynamic light scattering instruments for the analysis of protein solutions, promiscuous inhibitors, buffers or other products in solution. Since its inception in the early 1990's it has become an indispensable tool for biomolecular sizing and characterization.