Amino Acid Analysis

Enzyme Research Proteomics utilises the PITC method of Amino Acid Analysis. Samples (either free amino acids or hydrolysed proteins) are coupled with PITC to form phenylthiocarbamyl (PTC) amino acids and then separated using a dedicated reverse phase HPLC. Pre-column derivatization chemistry, although more sensitive than post-column derivatization chemistry in which amino acids react with a reagent such as ninhydrin after they elute from an ion exchange column, is subject to interference from the sample matrix making it important to consider your final purification methodology.

Analysis can be perfomed on virtually any type of sample, both free amino acids or components of preoteins following hydrolysis. Analysis of physiological materials, cell culture media, fermentation media, and proteins is routinely undertaken.

Amino acid analysis of proteins is performed on high purity material. Should the desired protein is present in a heterogenous mixture, ERP is able to offer its protein purification and electrophoresis service to purify the target protein prior to sequencing.

Quantities. Accuracy increases with quantity. The minimum quantity of protein required for hydrolysis and analysis is 1 microgram. The detection limit is approximately 0.2 ug.

Medium Request. Samples should be free of detergent, amine salts and lipids. PITC will derivatize primary and secondary amines, including amine groups found in common detergents and buffers used in protein purification. PTC-amine salts will co-elute with some amino acids and/or interfere with the derivatization chemistry and detergent will prevent the chromatography from working properly. Non-volatile amine salt buffers such as TRIS are incompatible with PITC amino acid analysis. Ammonia in the sample matrix should be minimized due to its reaction with PITC to form phenylthiourea which, in large amount, will block detection of certain amino acids.

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