Amino Acid Analysis - Amino Acid Quantification - Protein ID Services
Amino Acid Analysis Services
Microanalysis of Amino Acids
Physiological Analysis of Biological Fluids
Food Content Analysis
Amino Acid Testing Service Platform
We perform amino acid analysis using a Waters Breeze™ 2 HPLC System, Waters 1525 Binary HPLC pump, Waters 2475 Multi λ Fluorescence detector and Waters 717 plus Autosampler injector. This system performs reverse-phase chromatography by fluorescence detection. Prior to chromatography, pre-column derivatization of the amino groups is performed using AccQTag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) chemistry under aqueous conditions.
Amino acids are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC). Following the pre-column derivatization of the analytes, separation and detection
is achieved using a reversed-phase column and a multi wavelength fluorescence detector.The analysis allows for the identification and quantification of up to 42 amino acids and related compounds. These samples are automatically
analyzed with assured performance methods and reports are generated using pre-defined software templates.
The Waters AccQ•TagTM amino acid analysis system is used for derivatization
Customers retain all rights to the sequence data and related intellectual property.
Amino Acid Testing Sample Preparation Guidelines
It is advisable to contact Bio-Synthesis prior to sample submission to discuss the required analysis. This helps to ensure that the most efficient and cost-effective analytical methods are employed. A sample submission form and guidelines should accompany each sample set.
- Dry, In solution, PVDF membrane
For best results, samples should be supplied in MilliQ water, PBS, phosphate buffer or dried in a 10x75 mm rimless Pyrex tube. If your protein is not readily soluble, please inform us as this may greatly influence quantitation.
Dry protein/peptide samples
For quantitation, Bio-Synthesis recommends that the customer submits three 10 µg samples (30 µg total) at a concentration of approximately 0.5-2 µg/µl. This is ideal for accurate pipetting and minimal input of buffer to the sample. We know that this is not always possible so this is just a guideline. For quantities below 5 µg, please call first so that a plan can be made to assure accurate data. Although our fluorometric detectors possess sensitivity to picomole level, at low sample quantities, background contamination becomes significant, making interpretation of the data difficult.
For molecular weight between 1000-25,000 Dal: submit ~ 5.0 µg (>50
pmols) of protein sample.For molecular weight <1000 Dal: submit ~1.0 µg (~1,000 pmols)
Analysis of proteins blotted to PVDF may produce mixed results. Out of the several factors that influence the quality of the analysis, protein "concentration" is the most important. It is recommended that samples be greater than 1 µg and blotted to polyvinylidene fluoride (PVDF). Nitrocellulose and nylon membranes are not recommended. For best results, it is recommended that electroblotting be performed in a non-Tris or non-glycine-containing buffer. Ccustomers may stain electroblotted proteins with Coomassie Brilliant Blue R-250 or Ponceau S., excise the samples from the membrane using a razor blade, and then wash the excised membranes thoroughly, preferably by vortexing with HPLC grade water in a clean Eppendorf-style tube. As PVDF membranes may contain a number of contaminants, it would be advisable for customers to supply a blank piece of stained/destained PVDF membrane to act as a control sample. See blotting or staining protocols. It should be noted that tryptophan and cysteine cannot be directly quantitated. For samples submitted in liquid form, the amino acid quantities of tryptophan and cysteine can be obtained.
Feeds and Solid Samples
Solid samples can be HCl hydrolyzed directly. For best results, it is recommended that the sample be finely ground to insure homogeneous hydrolyzation.
Free forms of amino Acids
Free forms of amino acids can be quantitated. Please ensure that the matrix in which these amino acids are submitted be free of large amounts of proteins, lipids, and carbohydrates. The assay works well with serum which has been spun through a molecular weight 5000-6000 cutoff filter. Examples of free forms of amino acids that are routinely assayed are glutamine, asparagine, citrulline, B-alanine, taurine, tryptophan, and ornithine.
Amino Acid Analysis requires an adequate amount of purified sample for accurate composition and quantitative data analysis. Purification protocols could possibly contribute to sample contamination and loss, so it is important that a clean environment is maintained.
Tips to help reduce Amino Acid Sample Contamination.
- Most samples containing proteins, peptides, and free form amino acids can be analyzed.
- Avoid the presence of buffers, trace metals, detergents, and high salts. Salts can alter the pH of the sample, causing the derivatization to either be incomplete or unsuccessful. The recommended salt concentration is 0.1 M or less.
- Avoid the presence of amino-containing substances since they may react with the carbamate.
- Significant levels of Tris, HEPES, glycerol (over 5%), lipids and carbohydrates can be problematic. If you encounter difficulty in cleaning up your sample, try using other desalting methods such as precipitation or reverse phase HPLC cleanup.
- Avoid contaminating the sample with fingerprints and dust, these contain large amounts of human proteins that will obscure the result.
- 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.
Samples submitted for Quantitation:
High Sensitivity Amino Acid Analysis
Its good practice to supply pure proteins in solution. Please note the above mentioned conditions andprovide information on the approximate number of picomoles of the submitted sample so that the addition of internal standard can be determined.
Analysis from PVDF is not quantitative, however, it can
provide an estimate of the amount of protein present.
Quantitative Amino Acid Analysis
Its good practice to supply dry samples such as food products, protein, and synthetic peptides in sufficient amounts for accurate weight. At least 100 mg of food product is needed and several milligrams of synthetic peptides or protein samples.
- 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 contact us.
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.
Sample Submission and Ordering
- Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment. Store sample in Eppendorf Safe Lock tube packed in such a way that it's cushioned from the effects of handling. If a gasket-equipped tube is not available, use Parafilm to assure that the cap does not pop off in shipping. Keeping the sample cool is at the discretion of the investigator. It is not necessary to preserve biological activity or structural integrity of the sample unless solubility will become an issue. Dried samples and samples on PVDF do not require being kept cool.
- Alternatively, refrigerate to +4°C for cold shipment of the liquid sample.
Before sending a sample, please use the amino acid analysis submission form to alert us for the arrival of protein samples. Concurrently, a printed copy of the same amino acid analysis submittal form should accompany the sample. This form is available on our website and can be filled out on line.
Ship the sample to:
Attn: Amino Acid Analysis
612 E. Main Street
Lewisville, TX 75057
800.227.0627 | 972-420-8505