Introduction
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.
Introduction
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.
Sample Preparation
Proteins can by analyzed from PVDF membranes after electroblotting from 1D/2D gels.
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.
