N-terminal Edman sequencing

Get up to 30 N-terminal amino acids
Compare data to expected sequence
Fast turn-around and easy-to-understand report

Technical details, sample info, experts etc.

Edman degradation of your protein or antibody

N-terminal sequencing, often done by Edman degradation, is a requirement according to the ICH Q6B Guideline. It is a natural part of the structura...

Edman degradation of your protein or antibody

N-terminal sequencing, often done by Edman degradation, is a requirement according to the ICH Q6B Guideline. It is a natural part of the structural characterization of recombinant proteins used in clinical testing and to demonstrate comparability/consistency between cGMP batches.

Our automatic, cyclic Edman degradation procedure cleaves off the amino acid residue one at a time which allows us to identify each individual amino acid by chromatography. However, the analysis will not work for samples containing multiple N-terminals and for N-terminally blocked proteins.

Protein – n-terminal Edman sequencing service includes:

Optional 1D SDS gel electrophoresis of the protein and transfer to PVDF membrane
Sequencing by Edman degradation
Determination of up to 30 amino acids
Comparison of data to expected drug substance sequence

Antibody – sequencing service includes:

1D SDS PAGE separation of the heavy & light chain
Blotting onto PVDF membrane and staining
N-terminal Edman sequencing – up to 20 amino acids for each chain, running separately
Optional: De-blocking of pyroglutamic acid

Not exactly what you are looking for? Send us a request using the form below so we can adjust the analysis to meet your specific needs, or have a look at our other protein characterization services.

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Services

N-terminal Sequencing – 5 Residues

PVDF membrane bound or in solution proteins
N-terminal Edman degradation, determination of 5 amino acids
Reporting include raw data

$450.00

Results in 10-14 days

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N-terminal Sequencing – 10 Residues

PVDF membrane bound proteins
N-terminal Edman degradation, determination of 10 amino acids
Reporting includes raw data

$650.00

Results in 10-14 days

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Customized N-terminal Edman sequencing

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

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More information

We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

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Learn more in this application note and blog posts:

Application note

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Technical details

Introduction to Edman degradation

The Edman degradtion chemistry is a cyclic procedure: It couples the PITC reagent to the free N-terminal amino group and then cleaves off the protein. Next, the PITC coupled residue is transferred to a flask and converted to a PTH-residue. Finally, the HPLC chromatography identifies the amino acid. The Edman degradation reaction cycle is repeated until a sequence of N-terminal amino acids is determined.

Since the Edman sequencing process never is 100%, the signal intensity decreases as the process goes a long. Therefore, the amount of material needed also increases with increased number of residues.

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

Edman degradtion for n-terminal sequencing

N-terminal HPLC chromatogram distingushing all 20 amino acids except Cysteine

5 sequences of proteins sequencing by Edman degradation

Results from 5 cycles of N-terminal proteins sequencing by Edman degradation including a blank and a standard

Sample preparation

Sample requirements for Edman sequencing analysis

The analyses are performed on PVDF membrane bound proteins or pure in-solution samples.

Use this guide for help on How to convert your sample to PVDF membrane bound proteins. Please send the PVDF membrane samples as stained bands that are individually cut out from the membrane and placed separately in high-quality tubes. You can also ask us to run the 1D gel eletrophoresis and transfer to PVDF membrane.

Protein sequencing analysis by N-terminal Edmand degradation generally requires 30-50 picomoles of pure protein or 200-250 picomoles of antibody (if the antibody requires conversion to PVDF membrane).

If a protein sample contains more than one protein, the analysis shows a mixture of amino acids in each Edman degradation cycle, and characterization can become too complex to yield proper sequence data.

We use 1-d page electrophoresis for Edmand degradation

1D SDS PAGE and blot onto PVDF membrane before N-terminal protein sequencing

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Martin Villadsen

MSc in Engineering within Medical Biotechnology

Thanh Ha Nguyen

MSc in Biotechnology

N-terminal Sequencing – 10 Residues

N-terminal Edman sequencing

Edman degradation of your protein or antibody

Edman degradation of your protein or antibody