Instrumentation consists of a state-of-the-art Perkin-Elmer/Applied Biosystems Procise Protein Sequencing System. This is a highly sensitive system that can detect quantities as low as 1 picomole. It is also compatible with samples that have been electroblotted onto PVDF.
$200.00 set-up and first three cycles (3 amino acids)
$20.00 each additional amino acid
$5.00 Prosorb sample clean up (optional)
The Edman chemistry cycle consists of three stages:
The N-terminus of the protein couples with PITC under basic conditions to form a phenylthiocarbamyl (PTC)-polypeptide.
The peptide bond of the N-terminal PTC-residue undergoes acid cleavage from the polypeptide chain. This results in the release of an unstable anilinothiazolinone (ATZ) derivative of the amino acid.
The unstable ATZ-amino acid is converted into the corresponding phenylthiohydantoin (PTH) derivative. The PTH-amino acid is stable.
At the end of each cycle of Edman degradation, the PTH-amino acid is separated from reaction by-products and identified, by HPLC chromatography and UV absorbance. Although not part of the Edman degradation cycle per se, PTH-amino acid analysis, like Edman degradation, is an essential step in the protein sequencing process.
Sample Purity -- What to Avoid:
Sample purity is one of the most critical factors for successful protein sequencing. Samples should:
- Contain one protein component only.
- Be free of reagents which interfere with Edman degradation and the sequencing process (e.g., Tris, glycine, guanidine, glycerol, sucrose, ethanolamine, SDS, Triton X-100, Tween,, ammonium sulfate and other ammonium salts)
The two basic approaches that can be used to purify samples for protein sequencing are:
- SDS-PAGE and electroblotting
- ProSorb sample preparation cartridges (available in our lab)
SDS-PAGE and electroblotting should be used if the protein of interest is in a complex mixture of proteins. For proteins isolated in solution which are free of contaminating proteins but contain salts, buffers and/or detergents, use ProSorb cartridges.
Sample Preparation: Proteins/Peptides in solution:
For best results an optimal amount of to load on the sequencer is 200 pmole of peptide or protein.
Samples should be as homogeneous as possible and free of salts, detergents, lipids and other non-volatile ingredients (SDS, TRIS, etc.). Solutions used in dissolving samples should, therefore, contain only volatile components such as water, acetonitrile, etc. and be concentrated in a volume of no more than 60 ul. If your sample is supplied dried, it will be brought up in a 1:1:1 mixture of acetonitrile, water and acetic acid unless otherwise specified.
If your sample contains these non-volatile ingredients, we can pass it through a Prosorb column which will clean up your sample while at the same time deposit it onto a piece of PVDF. The fee for this service will be $5.00.
Sample Preparation: Electroblotted Proteins/Peptides:
For proteins/peptides adsorbed onto PVDF, again the optimal amount protein/peptide to load on the sequencer is 200 pmole. The piece of PVDF should be no larger than 3 x 7 mm and the number of pieces no more than five. Keep in mind that transfer is not 100% efficient so load an adequate amount of protein or peptide onto the gel prior to electrophoresis. Remember that the intensity of the stain is not indicative of sample amount and that the presence of a single band does not always indicate that there is only one species present.
Please note that nitrocellulose cannot be used in our automated sequencer.
If you would like to have a more detailed description of sample preparation, please look at the Perkin-Elmer/Applied Biosystems Guide to Sample Preparation.