Sample Preparation

Amino acid analysis using today’s instrumentation is a very sensitive assay. Sensitivity is, however, dependent on the absence of contaminants in the sample matrix. From our end, the hydrolysis tubes are pyrolyzed from 4-8 hrs at 400-500°C before use to remove residual amino acids and are handled wearing non-latex gloves and using clean forceps. We recommend that our clients use only high-grade chemicals and reagents

• Non-Volatile Salts & Buffers: Low levels of salts (<50 g of 0.5N NaCl) are acceptable. Non-volatile compounds, like glycerol, must be removed.
• Non-Volatile Amines: Buffers containing primary and secondary amines, particularly TRIS and other detergents, must be removed from the sample matrix. When derivatized, they will appear as undocumented extra peaks in the chromatogram and may be large enough to obscure a large portion of the chromatogram. The presence of the amines in these buffers will compete with the sample for the available PITC reagent and affect the quantitative derivatization of the amino acids in the sample.
• Lipids & Oils: Lipids, fatty acids, and oily substances must be removed from the sample matrix. They affect the hydrolysis and dissolution of the derivatives in the diluent buffer. More importantly, their presence is very detrimental to the column and may eventually make the column unusable. Samples with > 3% Fatty Acids/Lipids should be defatted and dried before submission. We may be able to assist with sample defatting if needed for an extra fee per sample.

Sample Preparation

• Liquid and Dried Samples: Ideally, we require 500 picomoles of purified protein, with an absolute minimum of 50 picomoles of purified protein. The purified protein may be dissolved in HPLC grade water or a volatile buffer/solvent, free of large amounts of the chemicals listed above under Sample Precautions. Dried or precipitated samples may also be submitted with documentation indicating compatible solvent(s).
• Electroblotted Samples: We can also conduct amino acid analysis from proteins electroblotted onto a hydrophobic membrane. This analysis is only for the amino acid composition of common amino acids (excluding cysteine and tryptophan). Quantitation of protein material present in the membrane is not accurate since the amount of protein that is bound to the membrane is unknown. Prepare samples using electrophoresis protocols (SDS-PAGE, non-denaturing PAGE, 2-D PAGE) and load sample on 3-4 lanes. Electroblot to polyvinylidene fluoride (PVDF) membrane. DO NOT use nitrocellulose or nylon membranes. Perform electroblotting in non-Tris, non-glycine-containing buffers. Coomassie Brilliant Blue R-250 or Ponceau S can be used for staining. Seal the whole membrane in a plastic sheet after destaining. Photocopy or scan the membrane and indicate which band you have selected for the analysis. PVDF membranes contain some contaminants themselves and a blank piece will be analyzed and used as a subtractive control.
• Foodstuffs: Various types of solid samples can be hydrolyzed directly in liquid HCl to yield an amino acid profile of the material. Please contact us for details and requirements.
• Lyopholized Tissues: Dried tissues can be hydrolyzed directly in liquid HCl to give amino acid profiles of the tissues involved in an experiment or comparative study. Please contact us for details and requirements.
• Physiological Fluids: Free amino acids in serum, urine, CSF, and other physiological fluids can be quantitated. The matrix of these fluids contains a large amount of non-amino acid components that interfere with the analysis. A sample clean-up procedure is necessary and a cost for this step is charged per sample. Please contact us for the particulars.
• Unusual Amino Acids: We are willing to attempt assay development for unusual amino acids you may be interested in quantifying. Please contact us to inquire.