Characterization of CtsK-Heparin Binding

To characterize the binding of pro-CtsK and mature CtsK to heparin, ~50 μg of purified WT or mutant CtsK was applied to a 1 ml HiTrap heparin–Sepharose column (Cytiva Lifesciences) and eluted with a salt gradient from 150mM to 2M NaCl at pH 5.5 in 20 mM NaOAc buffer. The conductivity measurements at the peak of the elution were converted to the concentration of NaCl based on a standard curve.

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Zhang X., Luo Y., Hao H., Krahn J.M., Su G., Dutcher R., Xu Y., Liu J., Pedersen L.C, & Xu D. (2024). Heparan sulfate selectively inhibits the collagenase activity of cathepsin K. bioRxiv.

Publication Preprint 2024

HiTrap heparin–Sepharose column

Corresponding Organization : University at Buffalo, State University of New York

Other organizations : National Institutes of Health, National Institute of Environmental Health Sciences, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Type of CtsK (WT or mutant)

dependent variables

Binding of pro-CtsK and mature CtsK to heparin
Concentration of NaCl required to elute pro-CtsK and mature CtsK from the heparin column

control variables

Purified WT or mutant CtsK (approximately 50 μg)
Heparin-Sepharose column (1 ml)
Elution buffer (20 mM NaOAc, pH 5.5)
Salt gradient (150 mM to 2 M NaCl)

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