Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agaro...

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Bibliographic Details
Published in:Analytical biochemistry 2011-10, Vol.417 (1), p.50-56
Main Authors: Cowman, Mary K., Chen, Cherry C., Pandya, Monika, Yuan, Han, Ramkishun, Dianne, LoBello, Jaclyn, Bhilocha, Shardul, Russell-Puleri, Sparkle, Skendaj, Eraldi, Mijovic, Jovan, Jing, Wei
Format: Article
Language:English
Subjects:
Calibration
Densitometry
Electrophoresis
Electrophoresis, Agar Gel - methods
Heat degradation
Hyaluronan
Hyaluronic Acid - analysis
Hyaluronic Acid - chemistry
Molecular mass
Molecular Weight
Reference Standards
Reproducibility of Results
Solution viscosity
Temperature
Viscosity
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Summary:The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000kDa. A sample load of 2.5μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2011.05.023