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Five grams of HA (MW 132K, 12.5 mmol) was dissolved in 250 ml of PBS solution (pH 7.0) and mixed well until no aggregation was observed. Three molar excess amounts of EDC (37.5 mmol) and HOBt (37.5 mmol) were added and stirred for 2 h, and then cystamine dihydrochloride (37.5 mmol) was added dropwise and mixed overnight. The reaction mixture was dialyzed for 24 h to remove unreacted cystamine and EDC and HOBt coupling agents (MW cut-off: 3500). Cystamine conjugated HA was treated with five molar excess of DTT and stirred for 24 h to cleave the disulfide bond in the conjugated cystamine. Thiol functionalized HA (HA-SH) was dialyzed against deionized water at pH 3.0 for 2 days (MW cut-off: 20,000). The final product was lyophilized for 3 days and stored at −20 ¡ãC until use. Percent thiol modification was determined by the Elman's assay using L-cysteine as a standard. Rhodamine labeled HA-SH (MW 17K) was also prepared by conjugating an amine-reactive rhodamine dye to HA using the same conjugation chemistry as above.

[ Last edited by scncwxy on 2010-11-29 at 16:52 ]
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scncwxy

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