FLUORESCENT TAGGING AND HPLC OF ONE 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Excipient Removal To six 500-[unreadable]L aliquots of the sample was added 10 mg ammonium bicarbonate, followed by, after vortexing and complete dissolution, 2 mL EtOH/EtOAc containing 0.5 % AcOH. The samples were vortexed for 20 seconds and allowed to stand for 10 min. The precipitate was pelleted by centrifugation and the supernatant was decanted. The pellet was dispersed in 500 [unreadable]L ammonium bicarbonate and precipitated again with EtOH/EtOAc/AcOH. The precipitation was repeated a total of 4 times. The pellet was then dissolved in 100 [unreadable]L 1 M AcOH. Protein Concentration A 10-[unreadable]L portion of each protein solution was diluted to 100 [unreadable]L with 1 M AcOH and the absorbance was measured at 280 nm. Mild Acid Hydrolysis A 50-[unreadable]L portion of each sample was mixed with 50 [unreadable]L 0.5 M sodium bisulfate, vortexed, and heated to 80 [unreadable]C for 20 min. OPD Derivatization To each hydrolyzed sample, as well as to N-acetylneuraminic acid (NANA) standards (100 [unreadable]L), was added 100 [unreadable]L 15 mg/mL OPD in 0.25 M sodium bisulfate. The mixtures were heated to 80 [unreadable]C for 40 min. After cooling, 800 [unreadable]L HPLC solvent A (see below) was added and 100 [unreadable]L of the resulting solution injected onto HPLC. HPLC Column: Ultrasphere ODS (4.6 [unreadable] 150 mm, 5 [unreadable]m, Beckman Cat. No. 235330) Solvent A: 0.2 % (v/v) n-butylamine;0.5 % (v/v) phosphoric acid;1.0 % (v/v) tetrahydrofuran in nanopure water. Solvent B: 50 % Solvent A, 50 % acetonitrile Gradient: 8 % B for 15 min, then 95 % B for 10 min, then equilibrate at 8 % B for 10 min. Flow Rate: 1 ml/min Detection: Fluorescence;Ex: 230 nm;Em: 425 nm