Analytical method for determining relative chaperone activity using an ovalbumin-conjugated column

Perturbation of the Relative Contribution of Molecular Chaperones in the Endoplasmic Reticulum

“…Each protein was conjugated with Affi-Gel 10 beads (Bio-Rad) (Table ) and subsequently packed into a column to obtain the native-form column of each protein. Since the denaturation temperatures of myoglobin, RNase A, and β-lactoglobulin were determined to be 54, 55, and 58 °C, respectively (determined by the method described in ref ) under the conditions of this study, thermal denaturation (70 °C, 5 min) of the protein-conjugated beads before column packing provided the denatured-form column of each protein. The ER fraction extracted from Wistar rat livers (10-week-old, male) with appropriate solubilized conditions without EDTA (modified from ref ) was applied to these columns, and the elution order of the chaperones was analyzed by Western blotting.…”

“…Recently, we reported a facile analytical method for estimating relative chaperone affinity under physiological multichaperone conditions. 7 In this method, a chaperone mixture was subjected to chromatography on a column conjugated with a denatured client protein, and the elution positions of target chaperones were compared to determine the relative binding affinity of each chaperone toward the denatured client protein. Thus, this method can estimate the conditions of the protein folding system.…”