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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
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Edman degradation of your protein or antibody

N-terminal sequencing 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/consi...

Edman degradation of your protein or antibody

N-terminal sequencing 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 – sequencing service includes:

  • Optional 1D SDS PAGE 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 so we can adjust the analysis to meet your specific needs.

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The process:

  • 1 You provide us with the sample as PVDF membrane or pure protein in-solution
  • 2 If sample is in-solution and complex: Ask us to run 1D gel and blot to PVDF membrane
  • 3 We determine up to 30 N-terminal amino acids by Edman degradation
  • 4 You receive the report within 4-8 work days

More information:

See an example of how results are presented

Get sample report N-terminal sequencing by Edman degradation

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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:

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 degradation

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

 

5 sequences of Edman degradation

Results from 5 cycles of N-terminal 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 and transfer to PVDF membrane.

Protein sequencing analysis by N-terminal Edman 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 n-term sequencing

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

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Martin Villadsen

MSc in Engineering within Medical Biotechnology

Read more
Do you need help?
Thanh Ha Nguyen

MSc in Biotechnology

Read more

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