N-terminal Sequencing

The Edman sequencing reaction remains the optimal method for obtaining amine-terminal residues.

The sequencer has sensitivity of under 1pmol and provides an alternative to mass spectrometry.

Request Form: N-terminal Sequencing 

 

Frequently asked questions

How should samples be prepared from gels?

The protein should be electrophoretically transferred to a good quality (protein sequencing grade) PVDF membrane. Such membranes are available from Bio-Rad, Applied Biosystems, GE and others. The membrane should be stained with Coomassie Blue, and the target band must then be visible by eye. The entire membrane should be sent intact along with a photocopy indicating the band to be sequenced.  PI will excise the band.
Please ensure the blots are free of any particulate material.

PLEASE NOTE: this service cannot be used for proteins in SDS-gels.

Can samples be analysed from liquid form?

Yes, however the sample must be freeze dried in a micro-centrifuge tube prior to shipping.

Sample should be free of interfering buffers and salts. Buffers containing primary amines, such as TRIS and HEPES, should be removed, although low concentrations of SDS (up to 0.3%) and PBS can be tolerated.

# Note: complex samples containing more than one protein cannot be analysed by this process.

What amount of protein is required?

For a pure protein the sensitivity limit is approximately 1 picomole, however we recommend 10pmol to ensure a satisfactory result.

What if the protein is blocked at the N-terminus?

No sequence information will be obtained and the “no result” fee applied.  The only alternative is to attempt internal sequencing in which case mass spectrometry based analysis is used.

Can Cysteine be detected?

Cysteine without special modification can not be detected by N-terminal sequencing and will give a blank result. The sample has to be modified for detection of cysteine.

Note: Sample should be freeze-dried or in solution.
Please indicate if you require Cys analysis.

Two specific points to note include:

  • Protein band must be visible by Coomassie Blue staining.
  • The membrane should be sent intact, along with a photocopy indicating the band to be sequenced.

 

Please refer to the FAQs brochure for further answers.

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