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a volume of 25 ¦Ìl (0.6%) oxidized HA and 25 ¦Ìl (30 mM) t-BC in 1% aqueous trichloroacetic acid were well mixed and allowed to react in a disposable Eppendorf tube at room temperature. After 24 h, 0.5 ml of aqueous TNBS solution(6 mM, 0.1 M borate buffer, pH 8) was transferred into the eppendorf tube to react with the excess t-BC. The t-BC-TNBS reaction was allowed to react for 60 min at room temperature.
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