Bio rad molecular weight standard4/6/2024 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). 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. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. 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. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. 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.
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