 | Protein Sequencing (Edman Degradation) |
| Principles: | The protein is reacted with the Edman reagent (phenylisothiocyanate, PITC) which modifies the N-terminus and the side-chains of Lys residues. Under controlled conditions, the N-terminal residue is released and converted to the phenylthiohydantoin (PTH) derivative which is identified by HPLC. Successive cycles of chemistry provide the N-terminal sequence of the protein. Some eukaryotic proteins are blocked at the N-terminus by acetylation and are therefore unable to be sequenced directly by this method. N-termini may also become blocked by chemical reactions such as carbamylation.
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| Sample: | Protein from reverse-phase HPLC or electroblotted onto PVDF. We recommend blocking the protein's thiols with iodoacetic acid if Cys is to be detected directly. The appropriate amount of protein is 2 to 100 pmol.
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| Experiment: | Typically, we bind the protein to a PVDF membrane or TFA-treated filter to immobilise it. Sequencing is then performed automatically for the agreed number of cycles.
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| Instrumentation: | Applied Biosystems Procise Sequencer (model 492) attached to a model 140C HPLC system with a model 785A absorbance detector operated under the control of 610A software.
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| Data Analysis: | The primary sequence is deduced automatically and validated manually.
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| Results: | Chromatograms from each cycle and a report are provided, describing the analysis undertaken and the deduced sequence(s). The report is either provided as hardcopy or sent by email, as Word document.
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| Costing (-GST): |
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