Characterization of High Concentration Protein Solutions by Dynamic Light Scattering

By AZoNano

Table of Contents

Introduction
Experiment and Results
Conclusion
About Avid Nano

Introduction

Dynamic light scattering (DLS) is a common technique employed in the determination of size and distribution of molecules, particles and their aggregates in different solvent conditions. In the protein crystallography field, unwanted aggregation can often during crystallization, as the process requires raising the concentration of the protein solution.

It is not possible to detect these aggregates using some DLS systems because of excessive light scattering from the concentrated sample. This problem can be handled with the W130i DLS system from Avid Nano as it features an automatic, wide range optical attenuator, which enables protein sample measurements at a wide range of concentrations.

Experiment and Results

BSA protein (66kDa MW) in a phosphate buffer was prepared at different concentrations of 50, 75, and 100 mg/ml, and the prepared samples were analyzed using the W130i DLS system, i-Size software, and innovative 5 µl BladeCell sample cuvettes from Avid Nano.

All the samples generated distributions with tiny secondary aggregate peaks, a common feature for very high concentrations. However, for BSA, it is not severe enough to hinder crystal nucleation and growth.

The estimated molecular weight and measured size are within reach of the true values for BSA at 50 mg/ml. However, significant decrease in measured sizes and corresponding molecular weights was observed at concentrations of 75 and 100 mg/ml.

Figure 1. Molecular weight and size measured by DLS at three concentrations.

Table 1. Dynamic light scattering data for BSA protein solutions.

Experiment

Rad (nm)

Est. MW (kDa)

Intensity

Mass

50mg/ml BSA

3.66
102.76

69.6
- - -

91.2%
8.8%

100.0%

75mg/ml BSA

3.39
55.26

57.4
- - -

88.1%
11.9%

100.0%

100mg/ml BSA

3.02
42.71

43.2
- - -

85.0%
15.0%

100.0%

Conclusion

The observed size change may not be due to multiple scattering phenomena, but because of molecule-molecule interaction, as the preparations are optically transparent and the raw DLS data is of superior quality.

The W130i DLS system’s capability to automatically compensate for highly scattering samples enables it to take size and distribution measurements for proteins at concentrations far higher than is possible with other DLS systems. At very high concentrations, however, the measured size may be different from the true size because of molecule-molecule interaction.

About Avid Nano

Avid Nano produce the most exciting and innovative new dynamic light scattering products. Avid Nano pursue the highest levels of performance, know-how, technology, convenience and reliability for all our products, used in laboratory applications requiring fast, accurate DLS measurements in the nano-metre size range.

The W130i dynamic light scattering system is a breakthrough, high sensitivity instrument for size and molecular weight measurement of proteins, bio- molecules and sub-micron particles. The W130i combines ultimate measurement performance with an economical benchtop footprint, rugged durability and supreme user convenience. It's extraordinarily compact dimensions are the result of miniaturized 'diamond geometry' S.A.B.Re fibre optic technology.

This information has been sourced, reviewed and adapted from materials provided by Avid Nano.

For more information on this source, please visit Avid Nano.

Date Added: Feb 13, 2013 | Updated: Jun 11, 2013
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