Amino terminal sequencing is carried out on an Applied Biosystems sequencer equipped with an on-line HPLC system. The limit of detection is 10 pmol of an individual phenythiohydantoin amino acid.100 pmol is usually sufficient to sequence from 5 to 25 residues - with the quality of the data increasing with the amount of sample. The maximum sample load is about 200 pmol. 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. Samples may contain small amounts of SDS (less than the equivalent of 0.1 ml of 0.01% SDS). After blotting samples onto PVDF, they should be stained with Coomassie Blue (silver stains may not be used) and then destained extensively with at least 4 changes of destaining solvent followed by a final wash with ultrapure water to lower the very high concentrations of Tris, glycine and other gel and transfer buffers that otherwise will interfere with sequencing. Please note that samples in SDS polyacrylamide gels may not be directly loaded onto our Protein/Peptide Sequencers. The cycle charges for amino acid sequencing include HPLC identification of the resulting PTH-amino acids and a summary table of the estimated PTH-amino acid yields. There is a minimum 5 cycle charge for samples which fail to sequence or which contain less than 5 amino acids. On extended sequencer runs only those cycles that are requested and for which a reasonable identification can be made will be subject to charge. The resulting sequence also will be searched via the National Center for Biological Information's databases (i.e., BLAST search of the nr database) at no additional charge. In those instances when a BLAST search does not identify the protein, an additional search will be carried out the EST database