The Edman degradation chemistry is a cyclic procedure where the PITC reagent is coupled to the free N-terminal amino group and cleaved off the protein. The PITC coupled residue is transferred to a flask, converted to a PTH-residue and the amino acid is identified by HPLC chromatography. The Edman degradation reaction cycle is repeated and a sequence of N-terminal amino acids is determined.
Since the Edman sequencing process never is 100% there will be a decrease in signal intensity as the process goes a long. Therefore the amount of material needed will increase with increased number of residues.
The Edman sequencing analysis will not work if the N-terminal amino group is blocked for the PITC chemistry due to e.g. acetylation or pyroglutamic acid which is frequent for antibodies. If the sample contains more than one protein, the chromatography will show multiple amino acids and a unique sequence cannot be read.
Protein sequencing analysis by N-terminal Edman degradation requires 2-10 micrograms, or 10-50 picomoles, of a pure protein.
If a protein sample contains more than one protein, the analysis will show a mixture of amino acids in each Edman degradation cycle, and a sequence can not be determinated.
Proteins can by analyzed from PVDF membranes after electroblotting from 1D/2D gels.
Alternatively, purified proteins in solution can be sequenced.
PVDF membrane samples
Eletrophoresis and electroblotting
Run 1D or 2D gel
Blot onto a PVDF membrane – DO NOT use Nitrocellulose membranes!
Wear gloves and avoid dust (keratin) contamination of gel and membrane
Stain the PVDF membrane with Ponceau or Coomassie Blue
Coomassie Blue and Ponceau S work well in combination with N-terminal Edman Sequencing – DO NOT use silver staining!
Wash the PVDF membrane in water
IF glycine is used in the transfer buffer, the PVDF membrane should be washed carefully to remove the glycine
Learn here more about optimal PVDF membranes, blotting buffers and staining methods for Edman sequencing
Cut out the bands/spots of interest
Use a clean scalpel to cut out the protein bands. They should be clearly visible by Coomassie Blue/Ponceau staining to have sufficient protein amounts
Cut the protein band close to the edge to have a high protein concentration on a small membrane area
Cut and send 2-3 identical PVDF bands/spots if possible, for eventual back up analysis
Put the bands/spots into separate wells/tubes
Purity and sample amount
The chromatographic protein purity should be >90%
Avoid all detergents and keep buffer concentration at a minimum.
Tris, glycine, guanidine, glycerol, sucrose, ethanolamine, SDS, Triton, X-100, Tween and other detergents, ammonium sulfate and other ammonium salts are among the reagents which may interfere with Edman chemistry and should be avoided.
Avoid contamination of the sample with dust, i.e. human keratin
Minimum amount ~ 1-10 micrograms
Put the protein/peptide into microcentrifuge tubes
Lyophilize or speed vac the protein/peptide to a solid sample to ensure protein stability during shipment
Alternatively, freeze or refrigerate the liquid sample for cold shipment
If the sample requires special treatment to bring into solution (e.g. organic solvents, extensive ultrasonic bath etc.) please write in the comment field on sample submission form