By Cooper C., Packer N.

Proteome structures Ltd, North Ryde, Australia. Describes a number of amino acid research suggestions and the way each one strategy can be utilized to respond to particular biologic questions.

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Additional resources for Amino Acid Analysis Protocols (Methods in Molecular Biology Vol 159)

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The amino acid derivatives are denoted by single-letter code, cysteic acid as CYA, and cystine as Cs. FFDA indicates the reagent peak; the peaks without denotations are reagent-related, as shown in an independent chromatographic run of the reagent alone. 3. Chromatographic Analysis 1. Prime the HPLC system according to the manufacturer’s instructions with solvent A and solvent B. 2. Equilibrate the column and detector with 90% solvent A and 10% solvent B. 3. Bring samples for analysis to room temperature; dilute, if necessary, with solvent A, and inject 20 µL (50–1000 pmol) onto the column (see Note 7).

3. 1. Preparation of the Macrospin column (sample 50–150 µ L) 1. The macrospin columns should be prepared basically as suggested by the manufacturer (2), except that we have introduced several more washes to better equilibrate the columns. In brief, tap the column gently to recover the gel material at the bottom of the column. Hydrate the gel with 500 µL of HPLC-grade water for 15 min at room temperature (see Note 6) and then centrifuge the column for 4 min at about 2000g to equilibrate the column.

Vapor-Phase Protein Hydrolysis 1. Pyrex glass vials (25–50 × 5 mm) from Corning. 2. Screw-cap glass vials. 3. 6 N HCl from Pierce. 2. Derivatization Reaction 1. Amino acid standard solution H from Pierce. 2. L-Amino acid standard kit LAA21 from Sigma. 3. Marfey’s reagent from Pierce. Caution: Marfey’s reagent is a derivative of 1-fluoro-2,4-dinitrobenzene, a suspected carcinogen. Recommended precautions should be followed in its handling (15). 4. HPLC/spectroscopic grade triethylamine, methanol, acetone, and dimethyl sulfoxide (DMSO) from Fluka.

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