Sample preparation for Edman sequencing
If you plan or consider sequencing a protein by Edman sequencing, this page will give you a short guide as to what to do.
What data do you need?
If you want to know the identity of a protein, mass spectrometry is usually the preferred technique.
If you want sequence data for cloning, mass spectrometry sequencing is again usually the preferred technique.
If you want to identify a proteolysis site, Edman sequencing is usually the preferred technique.
If you want to now if expression of a protein starts where you expect, Edman sequencing is often the preferred technique.
If you want to know where a radioactive amino acid is located, Edman sequencing is the only technique available here.
Many proteins are blocked to Edman sequencing
Edman sequencing requires a free amino group at the N-terminal of the protein. Often it is modified by acetylation or other changes so the protein cannot be sequenced by Edman sequencing. This is one reason mass spectrometry is normally used to obtain protein sequence data.
If you know something about your protein, you may be able to study literature to see the chances that your protein has a blocked or free N-terminal.
If a protein has a blocked N-terminal the original scientific question is usually addressed with some other technique.
Sample preparation choices
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Run a gel and transfer to PVDF. Nitrocellulose cannot be used. Stain PVDF with Ponceau or Coomassie. On an image of the blot, mark the bands to be sequenced. Or cut out the bands, with no excess, unstained PVDF, so they should be no more than 1.5 mm wide, and place in clean 1.5 ml tubes. If the bands stain with Ponceau or stain well with Coomassie, there is probably enough protein.
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Have the protein in aqueous buffer, of known composition. The protein will be adsorbed on to PVDF.
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Purify the protein or peptide by reverse phase chromatography so that it is in a solution of acetonitrile, water, trifluoroacetic acid.
In all cases, give the samples a name that is unambiguous and will not easily separate from your sample. Complete a sample submission form
How much sample?
10 pmole of a good protein is enough to read sequence data comfortably. However, often the amount of protein which reaches the sequencer is much less than estimated which is why more is recommended. If you are running a gel, one way to get an estimate of the amount of protein is to run a known quantity of a standard protein.
If you have more than minimal quantity of protein and there is no data, we can probably answer the question of insufficient protein or blocked protein. With sufficient blocked protein, there will be a relatively constant pattern of amino acids.
Blotting from a gel
You must use PVDF. Nitrocellulose will dissovle in the sequencer and may require very expensive repairs. Two types of PVDF made specifically for sequencing are ProBlott from Applied Biosystems and Immobilon-Psq from Millipore. These two types are claimed to give better yields of proteins, especially for small proteins.
The blotting conditions can be whatever works well for your protein. Tris-glycine can be used; we can wash out glycine wiht 20% methanol so that it does not interfere with the sequencing.
The PVDF can be stained with Ponceau, or Coomassie, or other stains compatible with sequencing. Applied Biosystems recommends staining for no more than a minute with Coomassie to keep background down.
Impure samples
If there is more than one protein in a sample, the correct sequences cannot be identified confidently. Unless one protein is present at a much higher amount than the other, we cannot confidently say which of two or more sequences has a particular amino acid. However, if there is an expected sequence, we can see if the data matches it. This is why the expected sequence is requested.