By Cooper C., Packer N.
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Extra info for Amino Acid Analysis Protocols (Methods in Molecular Biology Vol 159)
5. 6 × 50 mm test tubes. Use borosilicate only. Pyrrolysis oven (500°C capability) for glass cleaning (see Note 1). Hydrolysis system for vapor phase hydrolysis. Constant boiling HCl. Phenol (crystalline or liquefied). 2. Derivatization 1. AQC dry powder. Store in a dry place, up to 6 mo in a desiccator. Available from Waters Corporation. 2. 8, with 5 mM calcium disodium ethylenediaminetetracetic acid (EDTA). 24 g of boric acid in a clean beaker. Add 100 mL of water, stir to dissolve. Add 187 mg of calcium disodium EDTA.
A chromatogram from 1 nmol of a hydrolysate of a recombinant glycoprotein containing both N-linked and O-linked sites is given in Fig. 6. Role of AAA in a Biotechnology Laboratory 29 24. Proteins that are highly glycosylated will have some residual amino sugars that will appear as a broad peak that elutes in the Ile-Leu-Nle region of the standard chromatogram. Increasing the hydrolysis time to 72 h will eliminate this peak. 25. Methionine residues can also be unintentionally S-alkylated, but this can be detected by the presence of trace levels of homoserine, a hydrolysis product of S-carboxymethylmethionine that elutes between Ser and Glx.
Although both have excitation maxima approx 248 nm, they have radically different emission maxima, with the AMQ near 520 nm, and the amino acid products at approx 395 nm. This shift in emission maximum allows the derivatization mixture to be injected onto the HPLC column without need for excess reagent removal. Since the initial publication, a number of papers have described various applications and extensions of the original method (7–11). Fluorescence detection permits highly sensitive analyses with detection limits ranging from 50–300 fmol for the normal hydrolyzate amino acids.
Amino Acid Analysis Protocols (Methods in Molecular Biology Vol 159) by Cooper C., Packer N.