N-Terminal Sequencing uses a chemical process based on the technique developed by Pehr Edman in the 1950's
N-terminal protein sequence information continues to play a
significant role in modern structural and molecular biology. N-terminal
sequencing (also called edman sequencing) is most commonly used to
identify unknown proteins, confirm protein identity and quality (often
for quality control of recombinant proteins), and identify protein
N-terminus and cleavage sites. Long sequences of 50 amino acids or more
are possible with this technique.
N-terminal sequencing utilises the well-established Edman degradative
chemistry, sequentially removing amino acid residues from the
N-terminus of the protein and identifying them by reversed phase HPLC.
Pure proteins (>90%) usually generate easily interpreted data, but
insufficiently purified protein mixtures may also provide useful data.
• Samples should contain 2 picomoles or more of the protein/peptide to
be sequenced. A minimum of 10 picomole should be supplied for gel
samples or if clean-up is required.
• Protein can be submitted dry, in solution, as an SDS or native polyacrylamide gel piece, or electroblotted to PVDF membrane.
• Nitrocellulose is not compatible with the Edman chemistry.
The sample will be reconstituted in 0.1% TFA/20% acetonitrile unless
specified otherwise. If there will be a solubility problem please
Sample should be free from interfering buffers, salts and detergents.
Buffers containing amines, such as Tris and glycine, and detergents
should be avoided. PBS can be tolerated. If interfering buffers/salts
are unavoidable it should still be possible to sequence the material
after clean up. We ask that a minimum of 10 picomole be supplied if
clean-up is required. An additional fee will be charged for clean-up.
Note: The N-terminus of proteins can react with formic acid to form a
formyl N-terminus, thus artificially blocking the protein. Also, if the
protein is to be extracted from SDS gels, urea used as an
electrophoresis reagent must be very pure and should have no cyanate
ions present as these will carbamylate the proteins and block them.
Cyanate ions form particularly at alkaline pH. Urea solutions used in
gel preparation or solubilisation should be deionised with ion-exchange
resin if not made from ultra high purity urea. Moreover, samples
containing urea should not be stored for long periods and should not be
heated in order to avoid carbamylation. Ampholines used to create pH
gradients can also contain amines and reduce the efficiency of
sequencing. APAF recommends IEF strips pre-prepared by Bio-Rad or GE
Healthcare as these do not present this problem.
Protein can be passively eluted from polyacrylamide in an overnight
procedure for an additional fee. Gels should be stained with Coomassie
Blue R-250 or G-250. Do not use silver stain.
Note: Passive elution is generally less efficient than electroblotting
and does not effectively extract most high molecular weight proteins. It
should only be used for well stained protein gel bands of at least
10pmol and is not recommended for proteins that are greater than 60kDa.
On PVDF Membrane
In general, best results are obtained using 2D gels blotted to PVDF. The
protein can be stained with Coomassie Blue R-250, Amido black, Ponceau S
or Sypro Ruby. Do not use silver stain. Other stains may be compatible
but please contact us prior to use.
Coomassie Blue G-250 detects protein by mass
(from 10-30 ng per band). A moderately intense spot of 10kDa containing
100ng protein will have a concentration of 10 picomole. At 100kDa a
protein of the same intensity will only contain 1 picomole.
• No sequence will be seen if the protein is N-terminally blocked either
naturally (e.g. by formyl, acetyl or pyroglutamyl groups) or
accidentally during isolation and storage (see above Note regarding in
• Cys will give a blank cycle unless reduced and alkylated. Acrylamide
is the recommended alkylating agent. Specify on request form how Cys was
• Post-translationally modified amino acid residues (e.g. glycosylated
or phosphorylated) will give a blank cycle upon sequencing.
Biological samples coming into APAF should be accompanied by
documentation of potential pathogenicity or pathogen free status
otherwise APAF will presume all samples from human and animal origin are
potential pathogens and will be treated accordingly.
How do I send samples from Overseas to APAF?
For guidelines and documentation required visit our