N-Terminal Amino Acid Sequencing
*** Note: This Service has been discontinued & is no longer available at Pinnacle ***
Edman N-terminal protein sequencing is a long-established technique used both for validating known / suspected sequences and for identifying unknowns. Despite being largely superseded by mass spectrometric analysis, it is still retained as an option for users requiring unambiguous N-terminal amino acid identification.
Examples
of such applications might include monitoring the genetic stability & proper processing of expressed clones, researchers working on non-genome sequenced organisms and confirmation of sequence in the screening of combinatorial peptide libraries.
One benefit of Edman analysis is that it can be used to ID stained protein bands directly off blots on PVDF membrane. A useful 'rule-of-thumb' is that provided the band is clearly visible with Coomassie blue [CB] stain, you'll stand a good chance of success.
Other Edman-compatible stains would include Amido black and Ponsceau red.
The procedure relies upon a free amino group at the N-terminal of the protein, hence a major disadvantage is that many eukaryotic proteins (60 - 80%) are blocked to Edman analysis through post-translational modifications such as N-acetylation. However there are techniques that can help circumvent this issue.
Sample Requirements: Samples need to be supplied in ~ug amounts and can be either blotted (and CB stained) onto PVDF membrane or delivered in (or dried from) a solution (preferably ~ 5 - 10ul) of volatile buffer such as ammonium acetate with optional organic solvent.
The guidelines for sample preparation and cleanliness detailed in protein MS analysis apply here also.
NOTE: With blots we only accept samples transferred onto PVDF membrane. Do not supply samples blotted onto nitrocellulose or nylon as these are not compatible.
Proteins that are found to be N-blocked are only ever charged at our minimum of 6 amino acids, no matter how many residues were requested.
If you are unsure about any aspect of sample submission or require further advice, feel free to contact the unit.
Costing, sample submission and ordering details are located here.
