By Cooper C., Packer N.
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Extra resources for Amino Acid Analysis Protocols (Methods in Molecular Biology Vol 159)
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). 4. Elute with gradient as described in Table 1 (see Note 8). Resolution of 2,4dinitrophenyl-5-L-alanine amide derivatives of 18 commonly occurring L-amino acids and of cysteic acid is achieved within 120 min (Fig. 2) (see Note 9). 5. Determine the response factor for each amino acid from the average peak area of standard amino acid chromatograms at 100, 250, 500, and 1000 pmol amounts.
Heinrickson, R. L. and Meredith, S. C. (1983) Amino acid analysis by reversephase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal. Biochem. 136, 65–74. 4. van Wandlen, C. and Cohen, S. A. (1997) Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures. J. Chromatog. A 763, 11–22. 5. , and Chibata, I. (1976) Biosynthesis of norvaline, norleucine and homoisoleucine in Serratia marcescens.
If your sample is small, you can minimize losses in the cartridge by adding 100 µL of buffer to the prewetted membrane prior to the addition of sample. The sample can be added directly to this buffer. 14. Samples or washes larger than 400 µL are accommodated by loading multiple aliquots into the reservoir. The PVDF membrane should not be allowed to dry between aliquots. 15. Based on a recent ABRF study on amino acid analysis (8), samples bound to PVDF membranes tend to have higher error rates (20%) than those analyzed free in solution (12%).
Amino Acid Analysis Protocols (Methods in Molecular Biology Vol 159) by Cooper C., Packer N.