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. 2004 Sep 11;343(4):819–831. doi: 10.1016/j.jmb.2004.08.071

Figure 3.

Figure 3

Limited proteolysis experiments on His-tagged rabies P. His-tagged P was purified from E. coli and digested for one hour at 37 °C with different proteases and analysed by SDS-16% PAGE. A, Digestion with trypsin. Lane 1, no trypsin. Lanes 2–7, trypsin/P-His ratios (w/w) of 1 : 8000, 1 : 4000, 1 : 2000, 1 : 1000, 1 : 500 and 1 : 250. The reaction was quenched by adding PMSF (10 mM final concentration). B, Digestion with endoprotease Lys-C. Lane 1, no protease. Lanes 2–6, quantities of protease used per 1 μg of P-His in units (one unit hydrolyses 1 μmol of N-p-tosyl-Gly-Pro-Lys p-nitroanilide per minute at pH 7.7 at 25 °C). Lane 2, 3.75×10−5 units; lane 3, 7.5×10−5 units; lane 4, 1.5×10−4 units; lane 5, 3×10−4 units; lane 6, 6×10−4 units. The reaction was quenched by the addition of EDTA (5 mM final concentration). C, Digestion with thermolysin. Lanes 1–8, thermolysin:P-His ratios (w/w) of: lane 1, 1 : 64; lane 2, 1 : 32; lane 3, 1 : 16; lane 4, 1 : 8; lane 5, 1 : 4; lane 6, 1 : 2; lane 7, 1 : 1; lane 8, 2 : 1. The reaction was quenched by adding EDTA (5 mM final concentration). D, Western blot of P-His after digestion with trypsin (left) and thermolysin (right) using MAb 25C2. Lane 1, no trypsin. Lanes 2 and 3, trypsin/P ratios of 1 : 2000 and 1 : 8000, respectively. Lane 4, no thermolysin. Lanes 5 and 6, thermolysin/P ratios of 1 : 1 and 1 : 4, respectively.