SAX-HPLC OF 1 SAMPLE

University Of Georgia, Athens GA

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Abstract

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 ABC digestion Four separate 85 [unreadable]L aliquots of the 0.8 mg/mL biglycan PG solution were treated with 5 [unreadable]L 1 M NH4OAc buffer, pH 7, and 10 [unreadable]L chondroitinase ABC (F. heparinum, Sigma), AC (A. aurescens, Sigma), B (F. heparinum, Sigma) (1 U/mL in 50 mM TRIS/60 mM NaOAc buffer, pH 8), or water, and incubated at 37 [unreadable]C for 18 h. Three chondroitin sulfate A (CSA, bovine trachea, Sigma) standard samples (1.0, 0.1, and 0.01 mg/mL) were digested the same way (with chondroitinase ABC only). An additional negative control without biglycan was run as well. SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6[unreadable]250 mm Waters Spherisorb analytical column with 5 [unreadable]m particle size at 25 [unreadable]C. Analytes were detected by their UV absorbance at 232 nm using the following system. Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. Gradient: time (min) % B 0 3 10 25 30 40 55 100 56 3 The flow rate was 1.0 mL/min.