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Notes about Amino Acid Analysis at Vanderbilt

1. Provide approximate nmolar quantity to Eric Howard at the core: 6th floor of Light Hall.
2. Buffer-exchange out all the salts you can. Note down any remaining in the buffer.
   
3. Fill out request form with your grant and center numbers. Bring it with you.
   
4. Eric hydrolyzes your sample (a few ul) in a volume of 100ul, then injects a fraction of that onto his Beckman System Gold HPLC.
5. He frequently does 2 injections of different volumes, e.g. if the first one is off-scale.
   
6. It can take between 2 days and 2 weeks, depending on his workload (it's usually close to 2 days).
7. Results can be plugged into AAA.xls spreadsheet for analysis.
8. For "vol hydrolyzed", enter the total volume(ul) of the sample that you gave to Eric.
   
9. For "ratio applied", use: "Inject Vol ul" (from results sheet) divided by 100 (but check this with Eric)
10. For "pmol analyzed", enter the value in the "Quantity picomoles" column.
11. The amino acids are listed in the order they come off the column;  AsX is the sum of Asp+Asn; GlX is Glu+Gln
    (You can get the known composition from the ProtParam tool at the expasy site).
12. Look at the columns 'integer composition' and '% internal error' to get an idea of your sample's purity.
13. The bottom left corner of the worksheet shows how the outliers are calculated.
14. The bottom right gives: 'Total pmol Protein hydrolyzed', which is the amount of protein in the whole sample you gave to Eric.
15. The concentration then reflects your original sample's concentration, in pmol/ul, which is nmol/ml or micromolar.