|
|
|
Contact Information:
Molecular Biology-Proteomics Facility University of Oklahoma Health Sciences Center 975 N.E. 10th Street, BRC 1106 Oklahoma City, Oklahoma 73104 Phone: (405) 271-3921 Vist: Website |
Amino Acid AnalysisAmino acid analysis can be performed on aqueous solutions containing free amino acids or on proteins and peptides following hydrolysis to release the amino acids. Samples that require hydrolysis should be supplied dried in clean 10 x 75 mm glass culture tubes. Approximately 50-100 picomoles of protein are required for successful analysis. Two types of data can be obtained: 1) amino acid ratios or 2) quantitative determination of the total amount of each amino acid present in the sample. For quantitative determinations we must be supplied information on the approximate number of picomoles that are present in the sample so that we can determine the quantity of internal standard to be added. Samples should be free of any amines, TRIS buffer, or urea. Other salts are acceptable at low concentrations (less than 0.1 M in 100 microliters). Samples submitted for analysis of free amino acids (no hydrolysis) must be deproteinized prior to submission. A common method of deproteinization is protein precipitation by TCA (trichloroacetic acid) followed by ethyl acetate or ether extraction of the residual TCA. The standard method for protein hydrolysis uses 6N HCl in sealed evacuated tubes for 20-24 hrs. at 110o C. Tryptophan, glutamine and asparagine cannot be quantitated following this hydrolysis. Tryptophan recovery is variable between 0 and 40%, while glutamine and asparagine are completely converted to their respective acid forms. Amino acid analysis is performed by cation exchange chromatography. Amino acid elution is accomplished by using a two buffer system; initially eluting with 0.2 N sodium citrate, pH 3.28 followed by 1.0 N sodium citrate, pH 7.4. Amino acids are detected by on-line post column reaction with ninhydrin. Derivatized amino acids are quantitated by their absorption at 570nm wavelength, except for glutamic acid and proline, which are detected at 440nm wavelength. This procedure is performed on an automated Beckman system Gold HPLC amino acid analyzer. Order Forms Amino Acid Analysis order form (requires acrobat reader) Prices
|
 |
||
 University of Oklahoma Health Sciences Center Core Labs
|
