Amino Acid Analysis
***Effective June 28, 2013, the Keck Biotechnology Resource Laboratory will discontinue the following services: Amino Acid Analysis, Protein Sequencing (Edman Degradation), Large- and Small-Scale Peptide Synthesis. We are no longer accepting orders or sample submissions.
"The most accurate method of determining protein concentrations is probably acid hydrolysis followed by amino acid analysis. Most other methods are sensitive to the amino acid composition of the protein, and absolute concentrations cannot be obtained."(1)
In addition, this analysis can be used to confirm the amino acid composition of peptides and proteins, determine the ratio of proteins in a complex, the % incorporation of various (non-acid-labile) modifications, the amount of contaminating protein in DNA and other non-protein samples, and which amino acids are depleted or enriched in cell media and other fluids.
Amino acid analysis is carried out on a Hitachi L-8900 PH amino acid analyzer, usually following overnight in vacuo acid hydrolysis at 115°C to cleave proteins or peptides into free (single) amino acids. The analyzer uses an ion-exchange column with with a pH and temperature gradient to separate the amino acids, then continually reacts the eluant with ninhydrin for detection at 570 nm and 440 nm. The analyzer is calibrated during each set of samples with a 400 pmole mixture of amino acids and is operated via the manufacturer's programs and with the use of their buffers. EZChrome Elite (for Hitachi) software is used to run the analyzer and collect and analyze the data. The raw amino acid nmole data is then tabulated in a spreadsheet to calculate ug protein/peptide, mole percent , and the predicted number of residues of each amino acid.
(1) Waterborg, Jakob H. "The Lowry Method for Protein Quantitation", The Protein Protocols Handbook, 2nd Ed., pg 7.
