This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Chondroitinase digestion A solution (100 ?L total volume) containing 0.1 mg/mL sample, 50 mM NH4OAc buffer, pH 7, and 0.1 mU/mL Chondroitinase ABC (F. heparinum, Sigma) was incubated at 37 ?C for 24 h. The enzyme was inactivated by heating to 100 ?C for 2 min and the samples were centrifuged prior to HPLC analysis. SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6?250 mm Waters Spherisorb analytical column with 5 ?m particle size at 25 ?C. Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. The flow rate was 1.0 mL/min. Injection volume was 10 ?L. Detection was performed both by UV absorbance at 232 nm and by fluorescence after post-column derivatization. Regarding the latter, a 1:1 mixture of 0.25 M NaOH and 1 % 2-cyanoacetamide was added at 0.5 mL/min from a binary HPLC pump to the eluent from the column. The eluent was then heated to 120 ?C in a 10-m reaction coil, followed by cooling in a 50-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm. Commercial standard disaccharides (Dextra Laboratories) were used as standards for the chondroitinase digestion mixture.
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