Protein bound to the PVDF membrane is analysed by Edman sequencing procedure. One amino acid is cleaved off at the N-terminus of the protein, followed by ID of the amino acid by HPLC analysis. Repeated 10 times for ID of 10 amino acids. The analysis only provides good data for proteins that are not modified at the N-terminal. For many antibodies the N-terminal is blocked by Pyroglutamate. Report includes raw data.
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.
Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.
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A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:
If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.
This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:
If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!
The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:
My name is Anette and I am customer support scientist at Alphalyse. I make sure we take good care of your samples. If you need help, e.g. ordering or shipping samples, send me a message - I'm here to help.