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PMID: 21684248 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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

Analytical biochemistry ·Vol. 417 ·No. 1 ·2011-10-01 ·Pages 41-9

Bhilocha S, Amin R, Pandya M, Yuan H, Tank M, LoBello J, Shytuhina A, Wang W, Wisniewski HG, de la Motte C, Cowman MK

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.

MeSH Terms
Buffers Calibration Densitometry Electrophoresis, Agar Gel/methods Electrophoresis, Polyacrylamide Gel/methods Electrophoretic Mobility Shift Assay Humans Hyaluronic Acid/analysis,chemistry,isolation & purification Molecular Weight Reference Standards Reproducibility of Results Sepharose/chemistry
Chemicals
Buffers Hyaluronic Acid Sepharose
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Bhilocha Shardul
Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, Brooklyn, NY 11201, USA.
Amin Ripal
Pandya Monika
Yuan Han
Tank Mihir
LoBello Jaclyn
Shytuhina Anastasia
Wang Wenlan
Wisniewski Hans-Georg
de la Motte Carol
Cowman Mary K
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Article Info
Journal
Analytical biochemistry
Abbr.
Anal Biochem
ISSN
1096-0309
DOI
10.1016/j.ab.2011.05.026
Published
2011-10-01
Epub
2011-00-27
Pages
41-9
Language
English
Region
United States
NLM ID
0370535
PMCID
PMC3207642
Subset
IM
Grants
NICHD NIH HHS · R01 HD061918 · United States
NICHD NIH HHS · R01 HD061918-01 · United States
NICHD NIH HHS · R01 HD 061918-01 · United States
External Links
PubMed source DOI: 10.1016/j.ab.2011.05.026 PMC Full Text View journal details
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