Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan

Shardul Bhilocha, Amin Ripal, Monika Pandya, Han Yuan, Mihir Tank, Jaclyn LoBello, Anastasia Shytuhina, Wenlan Wang, Hans Georg Wisniewski, Carol De La Motte, Mary K. Cowman

Research output: Contribution to journalArticle

Abstract

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5-500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 μg (for polyacrylamide) to 2.5 μg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150-kDa HA standard.

Original languageEnglish (US)
Pages (from-to)41-49
Number of pages9
JournalAnalytical Biochemistry
Volume417
Issue number1
DOIs
StatePublished - Oct 2011

Keywords

  • Electrophoresis
  • Electrophoretic mobility shift assay
  • Glycosaminoglycan
  • Hyaluronan
  • Molecular mass
  • Molecular weight

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Bhilocha, S., Ripal, A., Pandya, M., Yuan, H., Tank, M., LoBello, J., Shytuhina, A., Wang, W., Wisniewski, H. G., De La Motte, C., & Cowman, M. K. (2011). Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan. Analytical Biochemistry, 417(1), 41-49. https://doi.org/10.1016/j.ab.2011.05.026