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Figure 7.
FIG. 7. Proteolytic digestion of the purified protein. A,
time course of proteolytic digestion of the purified protein
with the linker in the absence of SDS. Chymotrypsin was added to
a purified protein solution (0.3 mg/ml in 50 mM phosphate
buffer, pH 7, 1 mM EDTA) at a 1:10 ratio w/w, and digestion was
allowed to proceed for the following times: 30 min (lane 2), 1 h
(lane 3), 3 h (lane 4), and 5 h (lane 5). Lane 1, protein
control in the absence of protease; lane 6, protease control in
the absence of protein. B, time course of proteolytic digestion
in the presence of SDS. The digestion protocol is the same as in
A, except that the protein was incubated in the presence of 0.1%
SDS before adding the protease. Lane 1, protein control in the
absence of protease. Lanes 2-5, same times of digestion as the
corresponding lanes in A. In lane 6, the protein was boiled for
4 min in 0.1% SDS, cooled on ice for 1 min, and then the
protease was added. Digestion was subsequently carried out at 22
°C for 30 min (lane 7), 1 h (lane 8), and 3 h (lane 9).
Aliquots were taken at the corresponding times, and sample
buffer was added to give a 2% final SDS concentration; the
samples were frozen at -20 °C until electrophoresis. The
samples have not been boiled prior to loading in the gels. All
gels (15%) were stained with Coomassie Blue. Lane M, molecular
mass markers.
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