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

Edman sequencing of protein or antibody

N-terminal sequencing of protein is a requirement in the ICH Q6B Guideline and is often done by  Edman degradation. It is however also a natural part of the structura...

Edman sequencing of protein or antibody

N-terminal sequencing of protein is a requirement in the ICH Q6B Guideline and is often done by  Edman degradation. It is however also a natural part of the structural analysis of recombinant proteins – used in clinical testing and to demonstrate comparability/consistency between GMP batches.

Our automatic, cyclic Edman degradation procedure cleaves off the amino acid residue one at a time. This technique allows us to identify each individual amino acid by chromatography. Note, however, that 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 from the n-terminal by Edman
  • 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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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 sequencing
  • 4 You receive the report within 4-8 weeks

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Technical details

Introduction to Edman sequencing

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 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 electrophoresis and transfer to the 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 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

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Meet the experts

For this type of analysis our experts include:

Do you need help?
Martin Villadsen

MSc in Engineering within Medical Biotechnology

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Do you need help?
Thanh Ha Nguyen

MSc in Biotechnology

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