TY - JOUR
T1 - Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan
AU - Bhilocha, Shardul
AU - Ripal, Amin
AU - Pandya, Monika
AU - Yuan, Han
AU - Tank, Mihir
AU - LoBello, Jaclyn
AU - Shytuhina, Anastasia
AU - Wang, Wenlan
AU - Wisniewski, Hans Georg
AU - De La Motte, Carol
AU - Cowman, Mary K.
N1 - Funding Information:
This work was supported by the National Institutes of Health/National Institute of Child Health and Human Development ( NIH/NICHD, R01 HD 061918-01 ), the Othmer Institute for Interdisciplinary Studies of Polytechnic Institute of New York University (NYU), and the NYU–Poly Seed Grant program.
PY - 2011/10
Y1 - 2011/10
N2 - 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.
AB - 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.
KW - Electrophoresis
KW - Electrophoretic mobility shift assay
KW - Glycosaminoglycan
KW - Hyaluronan
KW - Molecular mass
KW - Molecular weight
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U2 - 10.1016/j.ab.2011.05.026
DO - 10.1016/j.ab.2011.05.026
M3 - Article
C2 - 21684248
AN - SCOPUS:80655147810
SN - 0003-2697
VL - 417
SP - 41
EP - 49
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -