Sample Submission
Protein sequencing can be a very sensitive method. However, its
sensitivity and usefulness are very dependent upon the quality of
the sample preparation as well as the amount of sample submitted.
Clearly, a sample contaminated with other proteins becomes more
difficult to produce useful data as the sequence of the 'target' may
be obscured by the presence of other sequences. In addition, too
little material makes determination of long stretches of sequence
less likely. The amount of material necessary for a successful
analysis depends, in part, on the physical medium in which the
sample is submitted.
Liquid Samples
Liquid samples between 10-20 pmol can be sequenced well
(depending upon the amino acids present). On the other hand, the
same protein submitted after electroblotting to PVDF membrane may
require 50-100 pmol to obtain the same amount of sequence. The
unique bi-phasic sample loading technology available from Hewlett
Packard offers a great deal of flexibility in sample buffers. The
sample may be submitted in non-volatile salts and buffers,
chaotropes, glycerol, organic solvents and small amounts of
detergents (please contact us). In addition, the sample may be
submitted in volumes up to 5 ml.
Electroblotted samples
Alternatively, samples may be sequenced directly from PVDF
membranes (nitrocellulose is unacceptable as it is not resistant to
the Edman chemistry). We have recommended protocols for
electroblotting and staining (please contact us before planning your
experiment). The sample cartridges can hold a considerable amount of
membrane material (60-80 mm2). This is roughly equivalent
to a slice 1-2 mm high and the entire width of a single mini-gel.
Most protein stains are acceptable (Coomassie, Ponceau S, Amido
Black) however, we prefer that a non-glycine electroblot buffer be
used.
Sample Loading
Prior to sequencing, your sample will be loaded onto the
sequencer column using a HP G1000A Sample Prep Station. During this
process your sample was either bound to C18 resin (liquid samples)
or inserted into an empty column space (PVDF electroblotted
samples). Liquid samples, regardless of solvent or volume, were
loaded in 2% TFA. The column was subsequently washed to remove
solvents and dissolved salts prior to mounting into the HP G1000A.
Instrumentation /
Sample Submission /
Sample Preparation / Sequencing Methods /
Results / Data Evaluation
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