N-terminal Sequencing

Sample Preparation Guidelines

N-terminal Sequencing by Edman degradation requires  2-10 micrograms of a pure protein to obtain good data. If a protein sample contains a mixture of proteins, the analysis will show a mixture of amino acids in each cycle.

Sample Preparation

Proteins should be submitted  dry as spots/bands on PVDF membranes. Alternatively in liquid or lyophilized form.

PVDF membrane samples

1. Eletrophoresis and electroblotting

1. Run 1D or 2D gel
2. Blot onto a 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 the PVDF membrane in water
   3. IF glycine is used in the transfer buffer, the PVDF membrane should be washed carefully to remove the glycine
   4. Learn here more blotting buffers and staining methods
      
      

3. Cut out the bands/spots of interest

   1. 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
   2. Cut close to the protein edge to have a high protein concentration on a small membrane area
   3. Send 2-3 identical PVDF bands/spots if possible, for eventual back up analysis
      
      

4. Put the bands/spots into separate wells/tubes

   1. Use the microcentrifuge tube or the 96-Well Plate supplied with the PICK 'n POST kit or a suitable tube 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 +4 C for cold shipment of liquid
      If the sample requires special treatment to bring into solution (e.g. organic solvents, extensive ultrasonic bath etc.) please write in comment field on sample submission form

Sample Shipment

The sample is now ready for submission

1. Fill out the Online Sample Submission Form in 5 easy steps. Make sure to include a print of the form with the samples.
2. Ship the samples using a Courier company. We have included an Air Waybill in the Pick ‘n Post kit to be used for secure shipment with FedEx/DHL using Alphalyse account. 

Alternatively, you can use a courier company of your choice. Ship to: