Services and
Fees
Contact us for services not listed below.
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Services
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Cost/Sample
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Normal HCL hydrolysis and Analysis (no Cys or Trp)
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$150
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Buffer blank or unusual/modified amino acid standard
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$85
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Cysteine determination (PAO + HCL hydrolysis + analysis)
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$230
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Tryptophan determination (MSA hydrolysis + analysis)
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$228
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Analysis only ("free" amino acids)
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$150
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Hydrolysis only (acid dried off and sample returned)
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$50
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Proteomic Facility's
Major Core Equipment
- Applied Biosystems 494 Procise High-Throughput Protein Sequencer
- Applied Biosystems 494 cLC High Sensitivity Protein Sequencer
- Hitachi L-8800 physiological fluids amino acid analyzer
- Applied Biosystems 3100 genetic analyzers
- Dionex DX-500 for monosaccharide analysis
- Applied Biosystems Voyager Mass Spectrometry
- Water 717 and 2475 multi wavelength fluorescence UV Detection
system
- Shimadzu HPLC system
We perform amino acid analysis using Water's ACQ-Tag method to derivatize amino
acid followed by separating the derivatives using reverse-phase HPLC and quantify
the results by using fluorescent detection. Breeze Software is used to run the analyzer
to collect and analyze the data. The raw amino acid nmole data is then tabulated
in a spreadsheet to calculate ug protein/peptide, mole percent, and the predicted
number of residues of each amino acid.
Quality Control
Quality control is provided by utilizing appropriate internal and external standards
as recommended by instrument manufacturers. Amino acid compositional analysis is
further assessed using a synthetic peptide containing one of each 20 amino acids
typically found in proteins. All analysis conducted utilize the latest manufacturer's
analytical software packages.
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Applications
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Microanalysis of Amino Acids
- Amino Acid Profile (Tryptophan not included)
- Standard Protein and Peptide Hydrolysis
- Trypotophan Determination (MSA)
- Cysteine determination (PAO)
- Amino sugars
- Other specific amino acids
Physiological Analysis of Biological
Fluids
- Plasma, Free amino Acids
- Urine, Free amino acids
- Cerebrospinal Fluid and other biological fluid
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Food Content Analysis
- Complete Amino Acid Profile
- Amino Acid Profile without Tryptophan
- Cysteine, Methionine and Lysine +9
- Standard Protein Hydrolysate
- Methionine, Lysine or Hydroxyproline
- Cysteine & Methionine
- Tryptophan
- Free Supplemental Amino Acids
- Complete Supplemental Amino Acid Profile
- Available Lysine and Complete Amino Acid Profile
- Glutamine (peptides and Protein)
- HMB - methionine analogues
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Customers retain all rights to the sequence data and related
intellectual property.
Amino Acid
Analysis Sample Preparation Guidelines
Prior to sending samples, investigators are recommended to contact the facility
to discuss the required analysis. This is necessary 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.
Sample Format
- Dry, In solution, PVDF membrane
For best results, samples should be supplied in MilliQ water, PBS, phosphate buffer
or dried in 10x75 mm rimless Pyrex tube. If your protein is not readily soluble,
please inform us as this may greatly influence quantitation.
Sample Amount
1. Dry protein/peptide samples
For the quantitation, three replicates of 10 µg each (30 µg total) is the best scenario.
And if the sample is in a concentration of approximately 0.5-2 µg/µl this allows
accurate pipetting while not adding a lot of buffer to the sample. We know that
it is not always possible to submit this much sample and good results can be obtained
with less. If below 5 µg of sample is all that can be obtained, please call first
so that a plan can be made to assure useful data. With our fluorometric detector,
we are able see with a resolution in picomoles. However, background contamination
becomes more and more problematic the less sample to work with and makes interpretation
of the data difficult.
The general rule:
1. Molecular weight between 1000-25,000 : ~ 5.0 µg (~20
pmols) of protein sample
2. Molecular weight >1000: ~1.0 µg (~1,000 pmols),
2. Electroblotted Samples
When analyzing proteins blotted to PVDF, mixed results are obtained. Out of the
several factors that influence the quality of the compositions, protein "concentration"
is perhaps the most important. Tryptophan and cysteine cannot be quantitated from
PVDF. If these amino acid quantities are required, the sample should be submitted
in a liquid form. Samples (> 1 µg ) that are prepared by electrophoretic procedures
(SDS-PAGE, non-denaturing PAGE, 2-D PAGE) should be blotted to polyvinylidene fluoride
(PVDF); nitrocellulose and nylon membranes are not compatible with hydrolysis. Electroblotting
must be performed in non-Tris, non-glycine-containing buffers. The electroblotted
proteins can be stained with Coomassie Brilliant Blue R-250 or Ponceau S. After
destaining, the sample can be excised from the membrane using a razor blade and
washed extensively by vortexing with HPLC grade water in a clean Eppendorf-style
test tube. As PVDF membranes contain some number of contaminants themselves, it
is required that the client supply a blank piece of stained/destained PVDF membrane
to act as a subtractive background control.
See blotting
or staining protocols.
Many different types of solid samples can be acid hydrolyzed directly in the liquid
HCl. Samples should be ground finely to assure a homogeneous sampling. About 100
mg is a good sample size for this assay, however, the assay can be scaled down to
~5 mg samples. Tryptophan and cysteine are not determined in this liquid hydrolysis
assay.
4. Physiological (Free Amino Acids)
Free amino acids can be quantitated. The matrix in which they are submitted should
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. Some free amino acids that are routinely assayed are glutamine, asparagine,
citrulline, B-alanine, taurine, tryptophan and ornithine.
Sample Purity
Amino Acid Analysis requires an adequate purified sample amount for accurate composition
and quantitative data analysis. Unfortunately, even the purification protocols used
can contribute both to sample contamination and loss. It is important that samples
are prepared in the cleanest environment possible.
See tips to help reduce Amino Acid Sample Contamination.
1.
Most samples containing proteins, peptides, and free amino acids can be analyzed.
2.
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
simply to fail. Acceptable salt concentration should be less than 0.1 M in 100 microliters.
3.
Avoid presence of amino-containing substance since it will react with the carbamate,
adversely affecting results. A 10% protein contamination can make your results meaningless.
4.
Significant levels of Tris, HEPES, glycerol (over 5%), lipids, carbohydrates can
be problematic. The glycerol is nonvolatile and attracts moisture (acid); the carbohydrates
char, decomposing to ash and taking the sample with them. If it is impossible to
clean up your sample, an attempt can be made other desalting methods, such as precipitation
or reverse phase HPLC cleanup.
5.
Avoid contaminating the sample with fingerprints and dust, these contain large amounts
of human proteins that will obscure the result.
6.
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 for Quantitation
High Sensitivity Amino Acid Analysis
For quantitative determinations we must be supplied information on the approximate
number of picomoles that are present in the sample so that we can determine the
quantity of internal standard to be added. 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.
Caution: 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 100 mg 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 contact us.
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.
Pathogenicity
Biological samples coming into APAF should be accompanied by documentation of potential
pathogenicity or pathogen free status otherwise APAF will presume all samples from
human and animal origin are potential pathogens and will be treated accordingly.
Sample Submission
and Ordering
Sample Storage
- 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 a 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.
Sample Shipment
1.
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.
Bio-Synthesis Inc
Bioanalytical Laboratory
Attn: Amino Acid Analysis
612 E. Main Street
Lewisville, TX 75057
800.227.0627 | 972-420-8505