 At higher pmol quantities, Edman sequencing will often provide the exact amino acid sequence without ambiguity for a limited run of amino acids, 6-30 amino acids, usually taking 30-50 min per cycle of the sequencer. The maximum sample load is < 1 nmol, with the best results generally being obtained on samples in the 10-100 pmol range. Samples should be submitted on PVDF membranes (less than 15 mm2) or in less than 0.1 ml of a volatile solvent such as water, 5 mM ammonium bicarbonate, 0.05% trifluoroacetic acid (TFA), or 50% acetonitrile/0.05% TFA. Solution samples may contain small amounts of SDS (less than the equivalent of 0.1 ml of 0.01% SDS). Samples should be free of non-volatile primary amines.
Edman sequencing is carried out on an Applied Biosystems protein sequencer equipped with an on-line HPLC system.
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