Amino Acid Analysis

Amino Acid Analysis

Amino Acid Analysis(AAA) is a service offered by MBCF to to quantify protein and peptides,  or to determine the identity of proteins or peptides based on their amino acid composition.  AAA is one of the most accurate methods of determining concentration of protein or peptide solutions.  In general, the sample is hydrolyzed by 6N HCL at elevated temp, the resultant mixture of amino acids are derivatized with a fluorescent tag which is then separated by HPLC and quantified by comparing to a set of known Amino acid standards similarly processed.

For amino acid analysis of samples we ask for the following:

  1. Samples, which are in liquid form to be 20-100uM in at least 20uL of volume. We need at least 200pmoles of individual AA to accurately determine concentration. For example: if  it is a peptide containing one Gly then we would need at least 200pm of peptide.  If it is a larger protein containing 10 Gly then we would need at least 20pm.
  2. If samples are in solid form, we can dilute the sample but will do so with water (please include mass of sample if in solid form).
  3. Buffer conditions will vary, but generally we prefer for samples to be in water. If you have an unstable sample, which needs to be in buffer, please let us know what concentration of buffer you will be using and what kind of buffer so we can determine whether it can go on our system. Generally we like samples to be in buffers that do not contain free amines. These amines will compete with sample for derivitization reagent and may cause low signal.
  4. Please include Peptide/protein sequence.
  5. Please include Estimated concentration of your sample.
  6. Please include Estimated molecular weight of sample.
  7. The cost is $35/sample.
  8. Your information: Name, lab, address, phone number, and PeopleSoft number (internal) or Purchase Order (external).

Samples submitted for analysis by AAA will be hydrolyzed with 6N HCl.  This will destroy some amino acids and convert others to their acid form.

Tryptophan will be destroyed and not reported

Cysteine can be partially destroyed and will not typically be accurate

Asn, and Gln will be converted to their acid forms and reported as total Asx, Glx