2-DE Spot Amino Acid Analysis with 9-Fluorenylmethyl Chloroformate

Amino acid analysis is a powerful and sensitive technique for the determination of the amino acid composition of proteins. In the past, the automation of amino acid analysis was based on postcolumn ninhydrin analysis, e.g., the Beckman 6300. However, a demand for high sensitivity and high sample throughput has led to the development of precolumn derivatization of amino acids and subsequent separation by reverse phase-high performance liquid chromatography RP-(HPLC), e.g., GBC AMINOMATE system. Although the chromophoric or fluorogenic derivatives allow greater sensitivity (100 fmol to low pmol range of protein), protein hydrolysis remains the limiting factor to high sensitive amino acid analysis. At the 5- to 20-pmol level, various contaminants influence the accuracy of both the qualitative and quantitative aspects of the analysis. Therefore, correct procedure and careful manipulation are required to achieve a successful analysis.