An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution

Gee Lee Hong Gee Lee, Mary Cowman

Research output: Contribution to journalArticle

Abstract

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'- dibenzothiacarboxyanine). The recommended sample load is 7 μg. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 106. The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA- binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 106. Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 106. These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Original languageEnglish (US)
Pages (from-to)278-287
Number of pages10
JournalAnalytical Biochemistry
Volume219
Issue number2
DOIs
StatePublished - 1994

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Hyaluronic Acid
Molecular weight distribution
Sepharose
Molecular Weight
Gels
Nylons
Membranes
Coloring Agents
Molecular weight
Aotidae
CD44 Antigens
Alcian Blue
Electrophoretic mobility
Silver Staining
Streptavidin
Synovial Fluid
Biotin
Knee Joint
Electrophoresis
Silver

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. / Hong Gee Lee, Gee Lee; Cowman, Mary.

In: Analytical Biochemistry, Vol. 219, No. 2, 1994, p. 278-287.

Research output: Contribution to journalArticle

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