Amino Acid Analysis

Liquid and Dried Samples

Amino acid analysis is a very sensitive assay. Unfortunately, with current instrumentation our ability to accurately quantitate the results depends more on the level of contamination present in the sample than on the amount of sample (see Sample Preparation). Therefore, ideally we request a minimum of 1 µgm of purified protein. The protein may be dissolved in a minimum volume of 100 µl of HPLC grade water or other pure volatile solvent that is free of large amounts of:

You may also submit dried or precipitated samples. In this case, be sure to include information as to what solvents are compatible with your sample. (Please see us to obtain the proper hydrolysis tube if you wish to dry your own samples for analysis.)

Electroblotted Samples

Alternatively, we can analyze amino acid composition from proteins that are electroblotted onto a hydrophobic support membrane. Tryptophan and cysteine cannot be quantitated from PVDF. If these amino acid quantities are required, the sample should be submitted in a liquid form. Samples (> 1µgm) that are prepared by electrophoretic procedures (SDS-PAGE, non-denaturing PAGE, 2-D PAGE) should be blotted to polyvinylidene fluoride (PVDF); nitrocellulose and nylon membranes are not compatible with hydrolysis. Electroblotting must be performed in non-Tris, non-glycine-containing buffers. The electroblotted proteins can be stained with Coomassie Brilliant Blue R-250 or Ponceau S. After destaining, the sample can be excised from the membrane using a razor blade and washed extensively by vortexing with HPLC grade water in a clean Eppendorf-style test tube. As PVDF membranes contain some number of contaminants themselves it is required that the client supply a blank piece of stained/destained PVDF membrane to act as a subtractive background control. If you need blotting or staining protocols, recipes or advice you may press HERE or call us at the PCL.

Feed and Flour Samples

Many different types of solid samples can be acid hydrolyzed directly in the liquid HCl. This assay is 'dirtier' than vapor phase hydrolysis requiring more sample (50 ugms) for accurate data. Tryptophan and cysteine are not determined in this liquid hydrolysis assay.

Free Amino Acids

Free amino acids can be quantitated. The matrix in which they are submitted should be free of large amounts of proteins, lipids and carbohydrates. The assay works well with serum which has been spun through a molecular weight 5000-6000 cutoff filter. Some free amino acids that are routinely assayed are glutamine, asparagine, citrulline, B-alanine, taurine, tryptophan and ornithine.

We are willing to try to develop assays for unusual amino acids and have some standard protocols on hand. Please drop in and discuss your particular needs.