Amino Acid Analysis technical background information
High-sensitivity amino acid analysis of protein
hydrolysates is performed using an Agilent 1100 series
HPLC equipped with an auto sampler performing an automated two-step pre-column derivatisation with two different reagents, OPA (o-phthalaldehyde) for primary,
and FMOC (9-fluorenylmethylchloroformate) for secondary amino acids . In case of D,L separation , a pre-column OPA derivatization in combination with a chiral thiol compound (IBLC; iso-butyryl-L-cysteine) is used to obtain stereo selectivity.
Detection limits are around 10 pmol, however, for accurate results a minimum of 50 pmol of signal is recommended. Since only a part of the hydrolysate is analyzed, this implies that about 2.0 µg protein material will give the best results.
The protein/peptide, as pure as possible and free of amines (Tris!), excessive SDS and salts, can be
sent to us either as a dry sample or in solution. Accuracy will be improved if also an analysis of an un-hydrolyzed sample is ordered, for background correction of free amino acids present in the sample /
Also, we offer amino acid analysis of proteins present in a small piece of SDS-PAGE gel, using a special protein isolation and clean-up procedure. The quantity of protein in the piece of gel should be at least
3-4 µg. Gels with a maximum thickness of 0.5-0.75 mm should be used, and standard Coomassie Brilliant Blue staining procedures can be applied. This approach results in far more accurate results than analysis of samples obtained by electro-blotting onto PVDF. The highest accuracy is obtained, if a blank piece of
gel from the same gel is simultaneously analyzed for background correction.
Hydrolyses are performed with HCl in the gas-phase for 1.5 hours at 150oC under which conditions Tryptophan is being destroyed and Asparagine and Glutamine will be deamidated into Aspartic acid and Glutamic acid, respectively.
Cysteine or Cystine can only be quantified accurately if modified to a stable derivative (alkylation or oxidation). Certain less common occurring amino acids like hydroxyproline can be quantified; for others, please inquire.
Analysis of unhydrolyzed samples for the quantification of free amino acids, also include Trp, Gln and
Asn. Other derivatives may also be quantified (on request). A special procedure is available for the quantification of free pyroglutamic acid in the presence or absence of protein.
Delivery: approximately 2 weeks after acceptance of order for standard analyses (one week
more for proteins present in a piece of gel). Includes raw data and calculation of the
amino acid composition.
Options: quantification of Cysteine; other hydrolysis time(s); analysis of unhydrolyzed sample(s); analysis of protein present in a piece of SDS-PAGE gel; D,L quantification.
In order to get the best results, please fill in the sample information part of the order form. We would
be pleased to advise you on the best way of preparing your sample.