title: Analytical method for determining relative chaperone activity using an ovalbumin-conjugated column.
abstract: Investigating the relative efficiencies of molecular chaperones is important for understanding protein biosynthesis inside a cell. We developed an analytical method for estimating relative chaperone activity under physiological, multi-chaperone conditions using a protein-conjugated column. A chaperone mixture was subjected to chromatography on a column conjugated with denatured ovalbumin, and the elution positions of target chaperones were compared using western blotting to determine the relative affinity of each chaperone for the denatured protein. Because molecular chaperones should be eluted according to their strength of association with the denatured ovalbumin in the column, the elution position must accord with the chaperone activity and can be used as an indicator of relative chaperone activity. We found that the column procedure was effective in an assay of a mixture of calreticulin and BiP, the molecular chaperones in the endoplasmic reticulum; the assay showed that calreticulin associated with denatured ovalbumin more strongly than BiP.
authors.affiliation: Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hirano
authors.affiliation: Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan.
authors.forename: Ayami
authors.initials: A
authors.lastname: Imagawa
authors.affiliation: Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino-shi, Tokyo 180-8633, Japan. Electronic address: ktotani@st.seikei.ac.jp.
authors.forename: Kiichiro
authors.initials: K
authors.lastname: Totani
mesh_terms: D000818:Animals; D037282:Calreticulin / Q000737:chemistry; D002417:Cattle; D002623:Chemistry Techniques, Analytical; D002845:Chromatography; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry*; D018832:Molecular Chaperones / Q000032:analysis / Q000737:chemistry*; D010047:Ovalbumin / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D012709:Serum Albumin / Q000737:chemistry
chemical_list: D037282:Calreticulin; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012709:Serum Albumin; D010047:Ovalbumin
keywords: BiP; Calreticulin; Molecular chaperone; OVA-conjugated column; Relative activity