Technologies
***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. Small-Scale Peptide Synthesis is no longer accepting orders. Please contact us for the last dates for order or sample submission related to other services.
HCl Hydrolysis and Analysis (AAA)
Prior to analysis, the majority of samples are hydrolyzed in vacuo for 16 hours at 115°C in 100 µl of 6N HCl/0.2% phenol to digest the protein or peptide into free amino acids. The phenol is used to protect tyrosine from hypochlorite/chlorine radicals in the acid that would reduce the yield of that amino acid.
Cysteine Determination (PAO)
Accurate quantitation of cysteine can be obtained by oxidation with performic acid (PAO) prior to HCl hydrolysis. Performic acid converts cysteine and cystine to cysteic acid, which is stable during acid hydrolysis and elutes away from other amino acids in the chromatogram.
Tryptophan Determination (MSA)
Best quantitation of tryptophan is obtained by hydrolysis with methanesulfonic acid (MSA) instead of HCl. MSA is not volatile, so it must be neutralized with NaOH to pH 1.5, the equivalent of the 0.2N HCl loading buffer used for other analyses.
Free Amino Acids (no acid hydrolysis)
For some projects, the amino acids incorporated in proteins or peptides are not of interest. If submitted in suitable solvents, buffers, or media, these samples can be dried in a vacuum centrifuge and dissolved in loading buffer for direct analysis.
Hydrolysis Without Analysis
HCl hydrolysis may be requested to digest protein to free amino acids, then the sample returned to the submitter for their own analysis. This is usually done for stable amino acid modifications that make the amino acid elute outside the analyzer's detection window or for modifications that co-elute with other amino acids.
