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Gainst time. The mean values from three experiments are shown with S.E. (error bars). Circles, wild type; squares, I54A; triangles, L47Q/L55Q.FIGURE 2. Co-precipitation of 32 with DnaK and/or DnaJ. A, 32 (0.4 M) was incubated with DnaK (1.6 M) at 30 for 30 min and treated with anti- 32 serum. The precipitates formed were subjected to SDS-PAGE. 32 and DnaK were detected by CBB staining (supplemen
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Bers were as follows: (DnaK)- 32-(DnaJ2) (184 kDa), Fractions 25 and 26; DnaK- 32 (101 kDa) and (DnaJ2)- 32 (114 kDa), Fractions 27 and 28; and DnaJ2 (82 kDa), Fractions 28 ?0. Because DnaJ was eluted over a wide range from the void volume to Fraction 30 (perhaps due to self-aggregation), the fraction numbers corresponding to a free DnaJ dimer (DnaJ2) were not precisely determined. Other Methods--
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Precipitated by adding protein A-Sepharose. After subjecting the precipitate to SDS-PAGE, 32 and DnaK were detected by CBB staining, and DnaJ was detected by immunoblotting with anti-DnaJ serum. Pull-down assays for the interaction between 32 and DnaKHis or DnaJ-His were performed using Ni2 -NTA-agarose. DnaK-His was preincubated in Buffer D (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 glycerol, 5 mM