All assays based upon a protein-dye binding reaction (Bradford, BCA, Lowry, etc.) are unreliable for determining the concentration of proteins. The values generated can differ greatly from the true protein concentration. These dye assays should be calibrated for each protein using AAA. This standardization will result in a much more reliable assay for determining the relative concentration of a protein. However, determination of the absolute concentration of the protein will still require AAA.
A data file comprised of a series of absorbance values vs. time points is generated during each chromatographic run. This file is stored on hard disk and is used by the software to generate a report identifying and quantifying each amino acid present.
The “Amino Acid Analysis Run Sheet” lists the run numbers, the corresponding sample ID’s and the sample volumes. In most cases, the sample will have been dissolved in Na citrate buffer containing 40 nmoles/ml Nle or in Li citrate buffer containing 100 nmoles/ml AEcys. The standard injection volume is 50.0 ul. Nle and AEcys are internal standards used to correct for any variation in the operating conditions of the analyser over time.
Each amino acid’s identification and quantitation is double-checked and underlined. Values for Pro (Asn and Hypro when present) are transferred from the 440nm printout. As the computer software can sometimes be “fooled”, there may be handwritten corrections on the printouts. All reports are reviewed by two staff members prior to release.
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