An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution
HG Lee, MK Cowman - Analytical biochemistry, 1994 - Elsevier
HG Lee, MK Cowman
Analytical biochemistry, 1994•ElsevierAn 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′-dibenzothiacarbocyanine). 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 …
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′-dibenzothiacarbocyanine). 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 …
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′-dibenzothiacarbocyanine). 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 × 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 × 106. Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 × 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.
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