Quantification of a protein or peptide

Protein or peptide quantification by amino acid analysis

You can use amino acid analysis to determine the:

  • Concentration of a purified protein when you know the known amino acid sequence.
  • Total protein amount in a complex sample with unknown proteins.
  • Accurate concentration on protein stock solution. You use it to calibrate other quantitative analyses, such as ELISA assays.

Amino acid analysis is the golden standard for accurate quantification of both proteins and peptides. The amino acid analysis process is quite simple: First a hydrolysis of an accurate amount of sample, and subsequently detection and quantification of individual amino acids. The analysis provides quantitative data for 18 out of 20 amino acids. However, degradation of tryptophan and cysteine occur during the hydrolysis.

Amino acid analysis:

  • Choose between a single or Triplicate amino acid assay. We recommend analysis in triplicates, whenever you need a very accurate protein or peptide concentration determination.
  • Comparison of results against your protein or peptide sequence.
  • Optional: It is very useful to combine the amino acid assay with an Extinction Coefficient measurement at 280 nm.

We analyze your samples with the newest technology and equipment in only few days. You get the results in an easy-to-understand report, also understandable to the non-expert.

If you need another type of amino acid analysis, send us your request for a custom project proposal.

Protein quantification by amino acid analysis

Quantification of Protein or Peptide

  • Hydrolysis of protein into free amino acids
  • Quantification of 18 amino acids, accuracy within +/- 10%
  • Calculation of µg protein amount (pmol if protein Mw is provided)


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Will be delivered in 4-8 days
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Protein quantification by amino acid analysis

Quantification of Protein Traces

  • Proteins at low concentrations
  • Hydrolysis of sample, quantification of 18 amino acids
  • Calculation of total protein content


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Protein quantification by amino acid analysis

Accurate Quantification of Protein or Peptide

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


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Will be delivered in 10-20 days
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Amino Acid Analysis Details

This amino acid assay is a method for quantifying proteins and peptides. We quantify a total of sixteen amino acids. These are the normal 20 amino acids except tryptophan and cysteine. Note that we determine asparagine as aspartic acid, and glutamine as glutamic acid.

The analysis process includes:

  • Protein hydrolysis
  • HPLC analysis
  • Data calculation

We base the Alphalyse procedure on the Ph. Eur and USP.


We perform the analysis on a BioChrom 30 amino acid analyser. It makes ion-exchange chromatography with post-column derivatization using ninhydrin. We can thus monitor all amino acids except Proline (440 nm) at 570 nm.

Protein hydrolysis – Vapour phase

First, we place the sample in a 0.5 mL hydrolysis tube and dry it down. We then place the tube in a vessel with an acid hydrolysis solution (6M HCl containing, 0.1% phenol, 0.1% thioglycolic acid). The vessel is flushed with argon. After evacuation (<10 mBar) the vessel is placed at 110°C for 24 hours. After hydrolysis the test sample is dried in vacuum. Next, we dissolve the sample in loading buffer (sodium citrate pH 2.20) including a fixed amount of sarcosine (Sar). We use this as an internal standard to correct for changes in reagent stability and flow rates. However, it also makes sure that the variation for each sample does not exceed 5%.

Calibration of Instrument

We analyze a standard mixture of amino acids at 3 different concentrations levels (double determination). This shows the linearity within the measurement range (700 to 2800 pmol). Next, a plot shows the peak area versus the known concentration of each standard solution. It passes when the r-value of the calibration is 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. We use the results 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. Based on at least two runs, a we then calculate a compensation factor to correct for ninhydrin activity. We correct each amino acid in the sample by this compensation factor.

Unknown protein samples: If there is no protein sequence, we now calculate the total protein amount based on the amount of each amino acid (in pmol), multiplied by the mass of the given amino acid residue. Please note that since 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 we know the protein sequence, we then divide the measured AA amount in pmol by the number of each specific amino acid in the sequence providing a pmol protein. We use the average of these numbers in pmol to calculate the amount of protein in µg with the molecular weight of the protein.

The calculations against known amino acid sequence is then made. We use a best linear fit based on all detected amino acids. It is modified for each experiment not to include amino acids with greater than 5 per cent variation.

Sample Preparation

You may submit samples in liquid form or as solid material.

Sample Purity

We can analyze most samples that contain proteins and peptides.
Avoid very high salt concentrations.
Do not use buffer that contain high amounts of primary amines, e.g. TRIS or glycine. Because it may interfere with the ninhydrin reaction and thus provide false quantification data of specific amino acids.
Avoid fingerprints and dust in the sample. 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 our facilities (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. Remember to print and include the sample submission form.

  • 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.
299 California Avenue, Ste 200
Palo Alto, CA 94306
Att: Thomas Kofoed
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
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. Remember to print and include the sample submission form.
  • 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. Remember to print and include the sample submission form.
  • 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
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