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Amino Acid Analysis Sample Preparation Guidelines

Amino Acid Analysis Sample Preparation Guidelines

Amino Acid Analysis determines the quantities of each individual amino acid in a protein, peptide or free amino acid sample. The Bio-Synthesis's Amino Acid Analysis service provides quantitative information for 18 out of 20 standard amino acids except tryptophan and cysteine which are destroyed during the analysis process. Asparagine is determined as aspartic acid and glutamine as glutamic acid.

Proteins and peptides are initially hydrolyzed using gas-phase HCl vapors in the presence of phenol and thioglycolic acid. The released amino acid residues are analyzed on an Accq-Tag Water amino acid analyzer using an ion-exchange column to separate the amino acids. Color is developed with ninhydrin. Free amino acid samples are analyzed directly without the hydrolysis step.

Results are delivered as raw chromatograms and in a report with table of picomoles and micrograms of each amino acid and total amount in microgram. As an option, customers can provide by email (biosyn@biosyn.com) specific protein/peptide sequence to be compared with the experimental data.

To read more about the Bio-Synthesis Amino Acid Analysis please visit us at www.biosyncom.

Samples can be submitted in liquid form or solid form

  1. Sample Preparation
  1. The sample should contain 5 micrograms or more of the protein to be analyzed.
  2. The sample can be submitted dry, in solution or blotted onto PVDF membrane.
  3. Purity of the sample is critical. The following types of contamination will interfere with the analysis:
    1.  Proteins, amino acids such as glycine and arginine, buffers and salts.
    2.  A 10% protein contamination can make your results meaningless.
    3. Tris, HEPES, glycerol and other primary and secondary amines must be avoided completely as this will obscure the amino acid analysis results
    4. Avoid contamination of the sample with fingerprints and dust, as they contain large amounts of human proteins.
  4. 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.
  5. 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.
  1. Sample 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
      • Tryptophan Analysis: Trytophan is destroyed by acid hydrolysis and requires a separate analysis 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 analyze samples that do not require hydrolysis.
  1. Place the sample into the microcentrifuge tube
  1. Use the Eppendorf Safe Lock tube.
  2. Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
  3. Alternatively, refrigerate to +4°C for cold shipment of the liquid sample
  1. Please indicate if:
  1. Download fill out Amino Acid Analysis Request Form
  2. Indicate if your primary objective is quantitation – for highest precision please supply protein sequence, accession number or expected composition, or
  3. Your primary objective is amino acid composition, or
  4. Your objective is both quantitation and composition

Sample Shipment

The sample is now ready for submission

  1. Download, fill out and sign the Amino Acid Sample Submission Form
  2. Place the tube(s) in the bubble bag to protect the tube during shipment.
  3. Ship the samples using a courier company. Liquid sample should be shipped at +4oC and gel samples at ambient temperature. Ship to:

    Bio-Synthesis Inc,
    Proteomic Analytical Division
    612 E. Main Street,
    Lewisville, TEXAS, 75057
    USA
    800.227.0627 (toll free)
    972.420.8505 (oversea)
    972.420.0442 (fax)