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Amino acid analysis is a fundamental biochemical technique used for the determination of the amino acid composition or content of proteins, peptides and other pharmaceutical or biological preparations or samples containing compounds that contain primary or secondary amino groups within their molecular structure. Amino acid analysis allows for amino acid quantitation (also known as amino acid quantification or amino acid identification) of free amino acids, as well as amino acids released from macromolecules such as peptides, proteins or glycoproteins. Amino acid testing also enables the analysis of protein complexes or mixtures of proteins such as protein powder supplements.
Protein molecules are abundant in mammals and are a significant and vital part of the mammalian diet as well as a vital part of their metabolism. Since proteins and various amino acids are needed in the human diet to help the body repair cells and synthesize new cells, amino acid quantification may be used to monitor or detect the metabolic states by analyzing the content of free amino acids in biological fluids such as urine, blood or plasma.
Bio-Synthesis follows GLP guidelines, offering comprehensive amino acid analysis and amino acid quantification services on biological compounds, foods, tissue, biological fluids, and drug samples. Using UV spectrophotometry, Bio-Synthesis can obtain accurate data on proteins, peptides, and amino acids of a particular sample. Protein characterization, concentration, content, molar ratio, and extinction coefficients can all be determined by using our amino acid analysis services.
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This is a standard analysis that recovers amino acids released from proteins. However, cysteine and trytophan have low probability of recovery (low amounts of trytophan may be recovered).
A buffer blank will be analyzed to determine if background content such as trace proteins, peptides, and amino acids is present or not.
In this analysis, proteins and peptides are first reduced in order to convert disulfide (-S-S-) bonds to sulfide (-SH) bonds. Then, the sample is treated with either iodoacetamide or vinylpyridine in order to maintain cysteine stability during the hydrolysis process. Finally, the modified cysteine is analyzed.
Bio-Synthesis accepts raw amino acid data for thorough analysis. The analysis results will be sent back to the customer with informative comments and suggestions.
Bio-Synthesis offers customers the option for only amino acid hydrolysis. The resulting hydrolysate will be dried off the acid and sent back to the customer.
Quality control is provided by utilizing appropriate internal and external standards as recommended by the instrument manufacturers. Amino acid compositional analysis is
further assessed using a synthetic peptide containing each of 20 amino acids typically found in proteins. All analysis conducted utilize the latest manufacturer's analytical software packages.
We perform amino acid analysis using Waters Breeze™ 2 HPLC System with software,a Waters 1525 Binary HPLC pump, a Waters 2475 Multi λ Fluorescence detector and a Waters 717 plus Autosampler injector. This system performs reversed-phase chromatography by fluorescence detection. Before the chromatography, pre-column derivatization of the amino groups is done using AccQTag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) chemistries 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
are achieved with a reversed-phase column and a multi wavelength fluorescence detector.The analysis is completed within approximately 45 minutes and 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.
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. Samples are normally analyzed in the order of their receipt, but special arrangements can be made for unstable samples. A sample submission form and guidelines should accompany each set of samples.
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.
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 the picomole , 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)
When analyzing proteins blotted to PVDF, we often obtain 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 use. For the best results, it is recommended that electroblotting be performed in a non-Tris or non-glycine-containing buffer. The customer can 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 potentially contain a number of contaminants themselves, it would be appreciated if the customer supplies a blank piece of stained/destained PVDF membrane to act as a background control. See blotting or staining protocols. It should be noted that tryptophan and cysteine cannot be directly quantitated. If the sample is submitted in liquid form, the amino acid quantities of tryptophan and cysteine can be obtained.
Solid samples can be HCl hydrolyzed directly. For the best results, it is recommended that the sample be finely ground to insure homogeneous hydrolyzation.
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 purified sample for a ccurate 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.
It would be appreciated if pure proteins are supplied in solution minding the above conditions, and information on the approximate number of picomoles of the sample is provided 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.
It would be appreciated if dry samples such as food products, protein, and synthetic peptides be supplied in sufficient amounts for accurate weight. At least 100 mg of food product is needed and several milligrams of synthetic peptides or protein samples.
Before sending a sample, please use the amino acid analysis submission form to alert us for the arrival of protein sample. Concurrently, a printed copy of the same amino acid analysis submittal form should accompany the sample. This form is available on this website and can be filled out on line.