A light scattering method for qualitatively monitoring aggregation rates in macromolecular systems

Bishop, John Bradford; Martin, J. C.; Rosenblum, William M.

doi:10.1016/0022-0248(91)90880-e

Cited by 20 publications

(7 citation statements)

References 3 publications

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“…Second, the polydispersity of the population is given by the standard deviation on D t . DLS is extremely sensitive to the presence of aggregates [15]. In structural biology, it is used to identify solutions in which macromolecules remain monodisperse during crystal nucleation and growth [16–21].…”

mentioning

confidence: 99%

Protein analysis by dynamic light scattering: Methods and techniques for students

Lorber¹,

Fischer²,

Bailly³

et al. 2012

Biochem Molecular Bio Educ

160

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Dynamic light scattering (DLS) analyses are routinely used in biology laboratories to detect aggregates in macromolecular solutions, to determine the size of proteins, nucleic acids, and complexes or to monitor the binding of ligands. This article is written for graduate and undergraduate students with access to DLS and for faculty members who wish to incorporate DLS into a lab activity, a practical course or research. It reviews the basic concepts of light scattering measurements and addresses four critical aspects of the analysis and interpretation of DLS results. To ensure reproducible quantitative data, attention should be paid to controlling the preparation and handling of proteins or assemblies because variations in the state of aggregation, induced by minor changes in experimental condition or technique, might compromise DLS results and affect protein activity. Variables like temperature, solvent viscosity, and inter-particle interactions may also influence particle size determination. Every point is illustrated by case studies, including a commercially available albumin, a small RNA virus isolated from plants, as well as four soluble proteins and a ribonucleoprotein assembly purified and characterized by students in the frame of their master degree.Keywords: Light scattering, protein, temperature, viscosity.During the last two decades the panel of methods used to study the structure and function of proteins and nucleic acids has grown continuously. An increasing number of instruments based on physical methods have appeared in biology laboratories. Amongst them, noninvasive spectroscopic methods based on light scattering have become a popular tool in biomolecule characterization. Light scattering measurements have numerous applications in condensed matter physics [1][2][3], biology [4][5][6][7], and medicine [8]. For half a century they have been used to monitor aggregation phenomena in protein solutions. Examples include the study of the influence of various factors on their state of aggregation [9], the detection and monitoring of undesirable aggregates in biotherapeutics that lead to immunogenic reactions or have adverse effects during administration in patients [10], and the investigation of protein aggregation diseases [11], such as caused by interactions between human gD-crystallin molecules with point mutations whose low solubility is responsible for lens opacity during cataract [12].Owing to intense monochromatic laser light sources, dynamic light scattering (DLS, also known as photon correlation spectroscopy or quasi-elastic light scattering) measurements provide a quick and straightforward means to determine the mutual translational diffusion coefficient (or diffusivity) D t of macromolecules in solution. D t describes the ease with which a substance displaces inside another one by diffusion and is equal to the mean square displacement of the particles divided by two-fold time [13]. According to Fick's first law of diffusion, D t relates the concentration gradient of a solute || To whom correspo...

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mentioning

confidence: 99%

Protein analysis by dynamic light scattering: Methods and techniques for students

Lorber¹,

Fischer²,

Bailly³

et al. 2012

Biochem Molecular Bio Educ

160

View full text Add to dashboard Cite

show abstract

“…This is likely to be related to the presence of a few larger entities (potentially agglomerates during the ultracentrifugation process). Since DLS is extremely sensitive to the presence of large entities, even with a low amount of these would account for the shape of the intensity-weighted data ( Bishop et al, 1991 ).…”

Section: Resultsmentioning

confidence: 99%

New Multiscale Characterization Methodology for Effective Determination of Isolation–Structure–Function Relationship of Extracellular Vesicles

Phan

Divakarla

Yeo

et al. 2021

Front. Bioeng. Biotechnol.

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Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

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“…Therefore, an analysis of the light scattering signal provides quantitative information about the behaviour, in solution, of the dissolved protein. In addition, if the intensity of the scattered light depends only on the spatial arrangement of the atoms, it is referred as Rayleigh-Gans-Debye scattering (Bishop, Martin, & Rosenblum, 1991). A typical DLS experiment takes just a few minutes.…”

Section: Protein Size Distributionmentioning

confidence: 99%

Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability

Oliver-Salvador¹,

Lian

Laursen

et al. 2011

Food Chemistry

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A light scattering method for qualitatively monitoring aggregation rates in macromolecular systems

Cited by 20 publications

References 3 publications

Protein analysis by dynamic light scattering: Methods and techniques for students

Protein analysis by dynamic light scattering: Methods and techniques for students

New Multiscale Characterization Methodology for Effective Determination of Isolation–Structure–Function Relationship of Extracellular Vesicles

Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability

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