N-Terminal sequencing

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.

For further details or advice on this APAF service please contact us at edman@proteome.org.au.

N-Terminal Sequencing Sample Preparation Guideline

Sample Preparation

• 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.

Dry Sample

 The sample will be reconstituted in 0.1% TFA/20% acetonitrile unless specified otherwise. If there will be a solubility problem please contact us.

In Solution

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.

In Gel

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.
Note: 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.
IMPORTANT:
• 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 solution samples).
• Cys will give a blank cycle unless reduced and alkylated. Acrylamide is the recommended alkylating agent. Specify on request form how Cys was modified.

• Post-translationally modified amino acid residues (e.g. glycosylated or phosphorylated) will give a blank cycle upon sequencing.

Pathogenicity

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 Quarantine guidelines.

 

Sample Request Forms


Download the N-Terminal Sequencing Request Form.  (PDF 341.7KB)