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Amino Acid Sequencing
Amino Acid Sequencing
Amino-terminal (Edman) sequencing is
carried out on an Applied Biosystems protein sequencer equipped with an
on-line HPLC system. The limit of detection on this high sensitivity
Procise 494 cLC instrument is about 50 fmol of an individual
phenylthio-hydantoin amino acid. In general, 1 to 10 pmol is sufficient
to sequence from 5 to 25 or more residues, with the quality of data and
length of sequence that can be assigned increasing with the amount of
sample. 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 not contain more
than the equivalent of 10 µl of 25 mM non-volatile salt and should be
free of non-volatile primary amines. Samples that have been
electroblotted onto PVDF should be stained with Coomassie Blue, Ponceau
S, or Immido black (silver stains may not be used), destained
extensively with at least 4 changes of destaining solvent, and washed
with 3-4 changes of ultra-pure water to lower the very high
concentrations of Tris, glycine, and other gel and transfer buffers
that otherwise will interfere with sequencing. After air drying, the
bands or spots of interest should be individually excised and placed in
1.5 ml Eppendorf tubes for shipment. 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 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 of the EST database.
(More
information)
Publications
Acknowledging the Keck Amino Acid Sequencing Resource
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