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Bjected to SDS-PAGE. The three standard proteins were detected by CBB staining, and DnaK (or DnaK-His), DnaJ-His, and 32 were detected by immunoblotting. Gel images were taken with LAS3000 image analyzer, and the quantitation of protein bands was performed using Multi Gauge software. The fraction numbers of the eluted proteins were as follows: -amylase (200 kDa), Fractions 25 and 26; apo-transferr
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D 10 l of a 50 slurry of Ni2 -NTA-agarose were then added, and the mixture was rotated at 4 for 1 h. Agarose beads were washed with Buffer F (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.2 Tween 20) five times, and DnaK-His and 32 were dissociated from the agarose beads by treating the precipitate with 250 mM imidazole. Proteins were subjected to SDS-PAGE and detected by CBB staining. Experiments u
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Wild-type at all temperatures (30, 37, and 42 ) (Fig. 1 and supplemental Fig. S2). These results indicate that stability of 32 in vivo does not always reflect its direct susceptibility to proteases and that the high stability of I54A 32 in vivo is attributed to some intracellular state of 32. Stable 32 Mutants Show Reduced Affinity for DnaJ--The above results showed that other factors in addition