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

Mary K. Cowmana, Cherry C. Chen, Monika Pandya, Han Yuan, Dianne Ramkishun, Jaclyn LoBello, Shardul Bhilocha, Sparkle Russell-Puleri, Eraldi Skendaj, Jovan Mijovic, Wei Jing

Research output: Contribution to journalArticle

Abstract

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 6000 kDa. A sample load of 2.5 μg 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 2000 kDa. 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.

Original languageEnglish (US)
Pages (from-to)50-56
Number of pages7
JournalAnalytical Biochemistry
Volume417
Issue number1
DOIs
StatePublished - Oct 2011

Keywords

  • Electrophoresis
  • Heat degradation
  • Hyaluronan
  • Molecular mass
  • Molecular weight
  • Solution viscosity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Cowmana, M. K., Chen, C. C., Pandya, M., Yuan, H., Ramkishun, D., LoBello, J., Bhilocha, S., Russell-Puleri, S., Skendaj, E., Mijovic, J., & Jing, W. (2011). Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan. Analytical Biochemistry, 417(1), 50-56. https://doi.org/10.1016/j.ab.2011.05.023