Amino Acid Analysis Sample Preparation Guidelines
Sample Preparation:
- The sample should contain 5 micrograms or more of the protein to be analysed.
- The sample can be submitted dry, in solution or blotted onto PVDF membrane.
- Purity of the sample is critical. The following types of contamination will interfere with the analysis: proteins, amino acids such as glycine and arginine, buffers and salts. A 10% protein contamination can make your results meaningless. Tris, HEPES, glycerol and other primary and secondary amines must be avoided completely.
- Low molecular weight solutes can be removed by dialysis (if you have sufficient protein), by reversed phase HPLC or by loading onto a ProSorb filter (PE Biosystems) and washing the PVDF membrane well with 0.1% TFA.
- For best results samples should be supplied in Milli-Q water, PBS, phosphate buffer or dried. If your protein is not readily soluble, please inform us as this may greatly influence quantitation.
Samples for Quantitation
High Sensitivity Amino Acid Analysis
Pure proteins are usually supplied in solution for quantitation and the above conditions apply. An aliquot will be taken and dried down before gas phase hydrolysis.
Note: Analysis from PVDF is not quantitative, however, it can provide an estimate of the amount of protein present.
Quantitative Amino Acid Analysis
Dry samples such as food products, protein and synthetic peptides need to be supplied in sufficient amounts to weigh accurately. At least 1g of food product is needed and several milligrams of synthetic peptides or protein samples. These undergo a liquid hydrolysis for accurate quantitation.
NOTE:
- Acid hydrolysis coverts asparagine and glutamine to aspartic and glutamic acid respectively. That is, the amino acid analysis result for Asp is a total of Asp + Asn and the result for Glu is Glu + Gln.
- For unusual amino acids,such as hydroxyproline, taurine, Norleucine and Hydroxylysine please inform us as this may greatly influence quantitation.
Tryptophan Analysis
Trytophan is destroyed by acid hydrolysis and requires a separate anaylsis using base hydrolysis.
Cysteine Analysis
Cysteine determination also requires a separate analysis. It is analyzed as cysteic acid after oxidation with performic acid.
Free Amino Acid Analysis
Free amino acid analysis is used to analyse samples that do not require hydrolysis.
Pathogenicity
Biological samples coming into our facility should be accompanied by documentation of potential pathogenicity or pathogen free status otherwise we will presume all samples from human and animal origin are potential pathogens and will be treated accordingly.
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