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Enzymatic/Chemical Cleavage of Proteins
Enzymatic/Chemical Cleavage of
Proteins
The majority of samples submitted for protein
identification/internal protein sequencing are submitted as
Coomassie Blue stained gel bands or spots that are then
subjected by the Keck Laboratory to in gel trypsin
digestion. The service charge for in gel digestion includes
carrying out a control digest on an otherwise identical
section of gel (supplied by the investigator) that does
not contain protein. The purpose of this control is
to enable rapid identification of reagent and trypsin
autolysis products. Upon request, a variety of other enzymes
(e.g., lysyl endopeptidase, chymotrypsin,
Staphylococcal protease, etc.) may be used in place of
trypsin. Although the Keck Laboratory will also carry out
enzymatic digestions on samples that have been blotted onto
PVDF, we strongly encourages investigators to avoid this
unnecessary step and instead, to prepare these samples
as
described and to submit them as Coomassie Blue (either
R250, G250 or "colloidal") stained gel bands. Although
proteins that have been stained with silver as described by
O'Connell
and Stults (1997) may be digested, our experience is
they may not digest as well as proteins that have been
stained with Coomassie Blue. The sensitivity of detection
with "colloidal blue" appears to be about 25 ng.
(More
Information)
Samples that do not require SDS PAGE purification may be
submitted dry (after vacuum centrifugation in a Speedvac) in
1.5 ml Eppendorf tubes that do not contain O-rings.
Although these samples generally may contain up to the
equivalent of 50 µl of 0.5 M non-volatile salts, the
investigator should email the details of these samples
(i.e., exact buffer composition and estimated protein
concentration) to the Protein
Chemistry Section prior to final sample preparation.
Unless requested otherwise, the Keck Laboratory generally
will reduce, carboxymethylate and then digest these samples
with trypsin in a final concentration of 2 M urea. In
addition to utilizing other proteolytic enzymes, samples
that are submitted for in solution digests may also be
subjected to cyanogen bromide cleavage upon request.
(More
Information)
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