Proteomlab beckman xl a centrifuge

AUC measurements were conducted with a ProteomLab Beckman XL-A centrifuge (Beckman Coulter, Brea, California) equipped with an AVIV fluorescence detection system (Aviv Inc., Lakewood, USA). For Hsp90 interaction studies, 500 nM labeled GR-LBD*, MR-LBD*, or Tau* (asterisk indicates labeled protein) were incubated with 10 μM unlabeled yeast Hsp90. p53-DBD* was used at a concentration of 1 μM. Wherever mentioned, nucleotides (ATP/ATPγS/AMP-PNP) were added at 2 mM concentration. For binding study with p23, 6 μM unlabeled yeast p23 was added to Hsp90 and client reaction mix in the presence of ATP. All the reactions were incubated for 2 hours at 25°C in 20 mM Hepes, 20 mM KCl, 5 mM MgCl₂, and 1 mM TCEP (pH 7.5) before measuring in AUC at 20°C. The reactions were prepared in a total of 320 μl of reaction volume. Data analysis was performed using SEDVIEW and OriginPro 9.1 (85 (link)). The fraction of labeled protein bound (f) was calculated from sedimentation curves as follows $$f=\frac{{\frac{\mathit{dc}}{\mathit{dt}}}_{\text{Norm},\text{free only}}-{\frac{\mathit{dc}}{\mathit{dt}}}_{\text{Norm},\text{free} \mathrm{w}/\text{complex}}}{{\frac{\mathit{dc}}{\mathit{dt}}}_{\text{Norm},\text{free only}}}$$ where ${\frac{\mathit{dc}}{\mathit{dt}}}_{\text{Norm},\text{free only}}$ means the normalized dc/dt value of free labeled protein in the absence of any addition; ${\frac{\mathit{dc}}{\mathit{dt}}}_{\text{Norm},\text{free} \mathrm{w}/\text{complex}}$ represents the normalized dc/dt value of free labeled protein left after complex formation in each case. The maximum value of dc/dt was used for each fitted curve.