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

•Denatured-ovalbumin (d-OVA) conjugated column was prepared.•The d-OVA column was effective for analysis of relative chaperone activity.•Relative activity of ER chaperones CRT and BiP was studied.•CRT associates with d-OVA more strongly than BiP. Investigating the relative efficiencies of molecular...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2015-01, Vol.456 (1), p.333-338
Main Authors: Hirano, Makoto, Kato, Yuki, Imagawa, Ayami, Totani, Kiichiro
Format: Article
Language:English
Subjects:
Animals
BiP
Calreticulin
Calreticulin - chemistry
Cattle
Chemistry Techniques, Analytical
Chromatography
Endoplasmic Reticulum - chemistry
Heat-Shock Proteins - analysis
Heat-Shock Proteins - chemistry
Molecular chaperone
Molecular Chaperones - analysis
Molecular Chaperones - chemistry
OVA-conjugated column
Ovalbumin - chemistry
Protein Binding
Protein Denaturation
Protein Folding
Relative activity
Serum Albumin - chemistry
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Summary:•Denatured-ovalbumin (d-OVA) conjugated column was prepared.•The d-OVA column was effective for analysis of relative chaperone activity.•Relative activity of ER chaperones CRT and BiP was studied.•CRT associates with d-OVA more strongly than BiP. 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.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2014.11.081