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Triplicate Amino Acid Analysis

for quantification of proteins and peptides (accuracy within +/- 5%)

Did you know that amino acid assay can be used to determine the:

  • Concentration of a purified protein with known amino acid sequence?
  • Total protein amount in a complex sample with unknown proteins?
  • Accurate concentration on protein stock solution to calibrate other quantitative analyses, as ELISA assays?

Amino acid analysis is considered to be the golden standard analysis for accurate quantitation of proteins and of peptides. As an example, we use the analysis to quantify the total protein amount in a complex sample. Another application is determination of the quantity of purified proteins and peptides.

We set up an amino acid assay consisting of:

  • Triplicate analysis for very accurate protein or peptide concentration determination.
  • Comparison of results against your protein or peptide sequence.
  • Optional: Combining the amino acid assay with an Extinction Coefficient measurement at 280 nm.

Our specialists analyse your samples with the newest technology and equipment. After completion of the data analysis they collect your results in a report, written in a language easily understood by the non-expert. As a last step, we email the final report to one or more persons of your choice.

If you just need a simple Amino Acid Analysis, consider our PICK ‘n POST quantification services. Or you can send us your request for a custom project proposal.

Services/Products
Protein analysis by Alphalyse

Accurate quantitation of purified protein by Amino acid analysis

  • Accurate quantification of purified protein, 3 replicates, accuracy +/- 5%
  • Comparison with protein amino acid sequence
  • Amount determined in picomoles & micrograms

$1,100.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

Extinction coefficient of protein biologics

  • Molar extinction coefficient determination
  • UV absorbance 280nm of protein dilution series
  • Triplicate amino acid analysis for exact concentration in dilution series

$2,200.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

Accurate quantification of total protein by Amino acid analysis

  • Accurate quantification of total protein amount, 3 replicates, accuracy +/- 5%
  • For mixtures of unknown proteins
  • Protein amount determined in micrograms

$1,100.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

Analysis Details

This Amino acid analysis refers to the method used to quantify proteins and peptides.

Sixteen amino acids are quantified – the normal 20 amino acids except tryptophan and cysteine. Asparagine is determined as aspartic acid and glutamine as glutamic acid. The analysis process includes:

  • Protein hydrolysis
  • HPLC analysis
  • Data calculation

The Alphalyse procedure is based on the Ph. Eur and USP.

Apparatus

The analysis is performed on a BioChrom 30 amino acid analyser by ion-exchange chromatography with post-column derivatization using ninhydrin. All amino acids except Proline (440 nm) are monitored at 570 nm.

Protein hydrolysis – Vapour phase

The sample is placed in a 0.5 mL hydrolysis tube and dried down. The tube is placed in a vessel containing an acid hydrolysis solution (6M HCl containing, 0.1% phenol, 0.1% thioglycolic acid). The vessel is flushed with argon and after evacuation (<10 mBar) the vessel is placed at 110°C for 24 hours. After hydrolysis the test sample is dried in vacuum and the sample is dissolved in loading buffer (sodium citrate pH 2.20) including a fixed amount of sarcosine (Sar). This is used as an internal standard to correct for changes in reagent stability and flow rates, and the variation for each sample should not exceed 5%.

Calibration of Instrument

A standard mixture of amino acids is analyzed at 3 different concentrations levels (double determination) to show linearity within the measurement range (700 to 2800 pmol). A plot is made showing peak area versus known concentration of each standard solution. The r-value of the calibration has to be better than 0.999 (proline is accepted at 0.995 due to lower signal/noise). The instrument is linear to 5600 pmol and usually up to ~20 nmol. Depending on the amino acid residue, the signal goes into saturation around 25-35 nmol.

Accuracy & Precision

For each batch run we include a duplicate analysis of a NIST BSA quality control standard. The results are used to determine the precision and the accuracy of the test. The precision should be below 5% and the accuracy within 15%.

Data calculations

A standard amino acid solution is analyzed for each 5 samples, and based on at least two runs, a compensation factor is calculated to correct for ninhydrin activity. Each amino acid in the sample is corrected by this compensation factor.

Unknown protein samples: If a protein sequence is not given, the total protein amount is calculated based on the amount of each amino acid (in pmol) multiplied by the mass of the given amino acid residue. Please note that as the content of cysteine and tryptophan is not included, the obtained value is underestimated by approximately 2.7% (average Cys and Trp content in proteins in the Swiss-Prot database).

Known protein samples: If the protein sequence is known, the measured AA amount in pmol is divided by the number of each specific amino acid in the sequence providing a pmol protein. The average of these numbers in pmol is used to calculate the amount of protein in µg using the molecular weight of the protein.

The calculations against known amino acid sequence is done using a best linear fit based on all detected amino acids, but will be modified for each experiment not to include amino acids with greater than 5 per cent variation.

Sample Preparation
Samples can be submitted in liquid form or as solid material.

Sample Purity
Most samples that contain proteins and peptides can be analyzed.
Avoid very high salt concentrations.
Buffer that contain high amounts of primary amines, e.g. TRIS or glycine, may interfere with the ninhydrin reaction and provide false quantification data of specific amino acids.
Avoid fingerprints and dust in the sample, as these contain large amounts of human proteins that will obscure the results

Sample Amount
Provide >100 microliters of liquid material at minimum concentration of 0.1 microgram per microliter.
Solid material should be provided in sufficient amount for accurate weighing of samples at Alphalyse (mg material)

Put the sample into the microcentrifuge tube
Lyophilize or speed vac the protein to ensure protein stability during shipment
Alternatively, refrigerate to +4°C for cold shipment of the liquid sample

Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork

Order analysis now
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