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Purified protein quantification

Quantification of pure proteins and peptides by amino acid analysis

We offer PICK ‘n POST™ quantification of purified protein & peptide samples

First, we hydrolyze the protein sample into free amino acids and separate them by ion exchange chromatography. Then, we derivatize with nihydrin, and quantify the amino acids. Finally, we determine 18 amino acids; these are the standard 20 amino acids except for tryptophan and cysteine, where we determine asparagine as aspartic acid and glutamine as glutamic acid. We also calculate the microgram protein quantity from the summed quantity of the individual amino acids. The picomole protein quantity is calculated with the estimated protein Mw you provide.

You can use this service for fast concentration determination of protein stock solutions.

You may want to have a look at our Biopharmaceutical Analysis services for more accurate triplicate amino acid analysis against a known protein sequence, and quantification of specific proteins in complex samples using LC MS/MS. We can also quantify the total protein or perform analysis of free amino acids already present in the sample. Look here for our other services for protein concentration determination.

This service includes:

  • You provide the protein and its estimated Mw.
  • We perform hydrolysis of the sample into free amino acids.
  • Ion exchange chromatography on an amino acid analyzer.
  • Quantification of 18 amino acids with accuracy within +/- 10%.
  • You receive the report within 4-8 days with the calculated protein content in micrograms and in picomoles.

How-to-order-PICK-n-POST

Examples of tables and graphs in report:

Services/Products
Fast and easy quantification of protein

PICK ‘n POST™ quantification of purified protein

  • Hydrolysis of protein into free amino acids
  • Quantification of 18 amino acids
  • Calculation of microgram protein amount
  • Calculation of picomole amount when protein Mw is provided
  • Accuracy within +/- 10%

$350.00

Fast and Easy
Will be delivered in 4-8 days
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Technical 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, the vessel is placed in a temperature monitored oven at 1100C overnight for 20 hrs. After hydrolysis, the sample tubes are dried in a vacuum centrifuge and the amino acids are re-dissolved in loading buffer.

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 requirements

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
Pipette 2-6 µg of liquid material into a 500 µl Eppendorf tube and lyophilize/speed vac it.
Alternatively provide 50-100 microliters (or more) 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

Sample shipment

shipping option
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
shipping option
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.

shipping option
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.
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.
  • 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.

shipping option
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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