| Bioactivity |
1. The reactions are in 33 mM Hepes pH7.2, 30 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 1.5 mM ATP in a 100 µl reaction at 37ºC. It is an enzyme-linked ATP regeneration assay tracking loss of NADH absorbance at 340nm. 2. The protein concentrations (where applicable) were 2 µM Hsp90 (SPR-102 Hsp90 beta), 2 µM Ahsa1 (His tagged human Ahsa1), 4 µM p23 (cleaved human p23), 4 µM HOP (His-tagged human HOP). 3. Addition of a two-fold excess of p23 reduced Aha1-mediated Hsp90 stimulation by 30% (when using 2 µM Hsp90 and Aha1). 4. Addition of a two -fold excess of HOP completely eliminated Aha1-mediated ATPase stimulation as well as intrinsic Hsp90 ATPase activity.
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