This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Heparinase digestionA 75 uL aliquot of a 1.5 g/L solution of low molecular weight heparin (AVT and HSP) in 20 mM ammonium acetate containing 2 mM calcium acetate was treated with 10 uL of a mixture of heparinases I, II, and III (1 U/mL each) and incubated at 37 C. After 20 h, the reaction was quenched by boiling the mixture for 2 min. ReductionA 60 uL portion of the heparinase-digested sample was treated with 10 uL of a 30 g/L solution of NaBH4 in 100 mM NaOAc buffer, pH 7, for 6 h at 23 C. The samples were then filtered (0.2 um) and injected into the SAX-HPLC system.SAX-HPLCSAX-HPLC was carried out on an Agilent system using a 4.6x250 mm Waters Spherisorb analytical column with 5um particle size. Analytes were detected by their UV absorbance at 232 nm. Two different 2-eluent systems were used to establish a salt gradient.
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