title: An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.
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'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. 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 10(6). 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 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). 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.
authors.affiliation: Department of Chemistry, Polytechnic University, Brooklyn, New York 11201.
authors.forename: H G
authors.initials: HG
authors.lastname: Lee
authors.forename: M K
authors.initials: MK
authors.lastname: Cowman
mesh_terms: D000818:Animals; D016645:Aotidae; D002232:Carbocyanines; D004396:Coloring Agents; D003720:Densitometry / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000302:isolation & purification; D007202:Indicators and Reagents; D008970:Molecular Weight; D013582:Synovial Fluid / Q000737:chemistry; D014822:Vitreous Body / Q000737:chemistry
chemical_list: D002232:Carbocyanines; D004396:Coloring Agents; D007202:Indicators and Reagents; C012040:stains-all; D006820:Hyaluronic Acid