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N-terminal Edman degradation

Determination of N-terminal amino acid sequencing

Want to know the N-terminal amino acid sequence of your drug substance protein?

We can list up to 40 amino acids of your drug substance. To do this, we use N-terminal Edman degradation. Our experts perform N-terminal sequencing, using the newest technology.

Edman degradation is a cyclic procedure. We cleave off the amino acid residues one at a time. Then we identify them by chromatography. If the sample holds 2-3 sequence variants the degradation can show this. However, if the sample contains multiple N-terminals it is not possible to read the aa sequence.

Following ICH Q6B? (Test Procedures and Acceptance Criteria for Biotechnological/Biological Products). ICH Q6B requires N-terminal sequencing of a drug substance.

Our N-terminal sequencing service includes:

  • Optional 1D SDS PAGE or HPLC isolation of the protein.
  • Sequencing by Edman degradation.
  • Determination of up to 40 amino acids.
  • Finally, a comparison to expected drug substance sequence.

We deliver the data in a report, which is easy to understand and use in your studies.

Download application note (pdf – 1 MB)

Not exactly what you are looking for? Then send us a request for a customized project proposal to meet your specific needs.

Need a faster and less expensive analysis? Take a look at our PICK ‘n POST Edman sequencing service. With this service you get the first 5, 10 or 20 amino acids analyzed in just 4-8 days!

Services/Products
Protein analysis by Alphalyse

N-terminal Edman sequencing of drug substance

  • N-terminal Edman sequencing
  • Determination of 10 amino acids
  • Comparison with DS sequence
  • From pure protein in-solution, lyophilized, or PVDF gel blot

$900.00

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

N-terminal Edman sequencing of drug substance incl 1D PAGE & PVDF blot

  • 1D PAGE separation, incl PVDF blot & staining
  • N-terminal Edman sequencing
  • Determination of 10 amino acids
  • Comparison with protein sequence

$1,750.00

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

N-terminal Edman sequencing of drug substance (customized)

  • Custom 1D PAGE, PVDF blot and band choice
  • Optional pGlu aminopeptidase deblocking
  • Edman sequencing, 10, 20 or more residues
  • Custom analysis for sequence variants, and semi-quantification

$2,000.00

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

Technical details

Introduction
The Edman degradation chemistry is a cyclic procedure where the PITC reagent is coupled to the free N-terminal amino group and cleaved off the protein. The PITC coupled residue is transferred to a flask, converted to a PTH-residue and the amino acid is identified by HPLC chromatography. The Edman degradation reaction cycle is repeated and a sequence of N-terminal amino acids is determined.

Since the Edman sequencing process never is 100% there will be a decrease in signal intensity as the process goes a long. Therefore the amount of material needed will increase with increased number of residues.

The Edman sequencing analysis will not work if the N-terminal amino group is blocked for the PITC chemistry due to e.g. acetylation or pyroglutamic acid which is frequent for antibodies. If the sample contains more than one protein, the chromatography will show multiple amino acids and a unique sequence cannot be read.

Sample requirements

Introduction
Protein sequencing analysis by N-terminal Edman degradation requires 2-10 micrograms, or 10-50 picomoles, of a pure protein.
If a protein sample contains more than one protein, the analysis will show a mixture of amino acids in each Edman degradation cycle, and a sequence can not be determinated.

Sample Preparation
Proteins can by analyzed from PVDF membranes after electroblotting from 1D/2D gels.
Alternatively, purified proteins in solution can be sequenced.

PVDF membrane samples
Eletrophoresis and electroblotting
Run 1D or 2D gel
Blot onto a PVDF membrane – DO NOT use Nitrocellulose membranes!
Wear gloves and avoid dust (keratin) contamination of gel and membrane

Stain the PVDF membrane with Ponceau or Coomassie Blue
Coomassie Blue and Ponceau S work well in combination with N-terminal Edman Sequencing – DO NOT use silver staining!
Wash the PVDF membrane in water
IF glycine is used in the transfer buffer, the PVDF membrane should be washed carefully to remove the glycine
Learn here more about optimal PVDF membranes, blotting buffers and staining methods for Edman sequencing

Cut out the bands/spots of interest
Use a clean scalpel to cut out the protein bands. They should be clearly visible by Coomassie Blue/Ponceau staining to have sufficient protein amounts
Cut the protein band close to the edge to have a high protein concentration on a small membrane area
Cut and send 2-3 identical PVDF bands/spots if possible, for eventual back up analysis

Put the bands/spots into separate wells/tubes

Liquid samples
Purity and sample amount
The chromatographic protein purity should be >90%
Avoid all detergents and keep buffer concentration at a minimum.
Tris, glycine, guanidine, glycerol, sucrose, ethanolamine, SDS, Triton, X-100, Tween and other detergents, ammonium sulfate and other ammonium salts are among the reagents which may interfere with Edman chemistry and should be avoided.
Avoid contamination of the sample with dust, i.e. human keratin
Minimum amount ~ 1-10 micrograms

Put the protein/peptide into microcentrifuge tubes
Lyophilize or speed vac the protein/peptide to a solid sample to ensure protein stability during shipment
Alternatively, freeze or refrigerate the liquid sample for cold shipment
If the sample requires special treatment to bring into solution (e.g. organic solvents, extensive ultrasonic bath etc.) please write in the comment field on sample submission form

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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