Protein Sequencing N-Terminal Amino Acid Sequence Edman Degradation

N-terminal Sequencing service determines the N-terminal amino acid sequence of proteins and peptides by the Edman procedure. Edman degradation is a cyclic procedure where PITC is coupled to the N-terminal amino group followed by cleavage of the N-terminal residue. The PITC coupled residue is transferred to a flask, converted to a PTH-residue and analysed by injection on an HPLC column.

Sample Preparation

Samples can be submitted in liquid form, solid form or as spots/bands from PVDF membranes.

PVDF membrane samples

1. Run your 1D/2D gel
   1. Run 1D or 2D gel
   2. Blot onto PVDF membrane – DO NOT use Nitrocellulose
      membranes!
   3. All handling should take place in a dust free environment
2. Stain the PVDF membrane with Coomassie Blue or Ponceau staining
   1. Coomassie Blue and Ponceau works well in combination with N-terminal Edman Sequencing – DO NOT use silver staining!
   2. Wash PVDF membrane several times in water
   3. Glycine in the transfer buffer requires very extensive washing and is therefore not recommended
3. Cut out the bands/spots of interest
   To be able to obtain good sequencing data you should only cut spots or bands that are clearly visible by Coomassie Blue/Ponceau staining
   1. Use a clean scalpel
   2. Cut out the band as close to the protein edge as possible to have the protein concentration high in the smallest membrane volume possible
   3. Clean the scalpel in-between spots/bands
   4. Please supply multiple identical PVDF bands/spots if possible (2-4 bands)
4. Put the bands/spots into separate wells/tubes
   1. Use the Eppendorf Safe Lock tube supplied with the kit or similar tubes from your lab

Liquid samples

1. Purity and sample amount
   1. The chromatographic protein purity should be >90%
   2. Avoid all detergents and keep buffer concentration at a minimum
   3. Avoid contamination of the sample with dust, i.e. human keratin
   4. Minimum amount ~ 1-10 micrograms
2. Put the protein/peptide into microcentrifuge tubes
   1. Use the Eppendorf Safe Lock tube supplied with the kit or similar tube from your lab
   2. Lyophilize or speed vac the protein/peptide to a solid sample to ensure protein stability during shipment
   3. Alternatively, refrigerate to +4oC for cold shipment of liquid
   4. If the sample requires special treatment to bring into solution (e.g.organic solvents, extensive ultrasonic bath etc.) please advise

Sample Shipment

1. Filled out sample submission form
2. Ship the samples using a courier company. Liquid sample should be shipped at +4^oC and gel samples at ambient temperature