(7) Assay Card

General Information
Summary Kalata B1 was found to be resistant to both chemical and thermal denaturation.
Condition CD measurements were carried out on a Jasco J-710 spectropolarimeter with a Neslab RTE-111 temperature control unit. Ellipticity changes at 220 nm were monitored to follow the unfolding transitions in the far-UV region (190-250 nm). All experiments were conducted in the range of 5-90 C. A path length of 1 mm was used, and all spectra were the average of three scans at a scan speed of 20 nm/min. Solutions of kB1 (50 uM) were prepared in 10 mM potassium phosphate (pH 2.5) in the presence and absence of 8 M urea.
Result Circular dichroism was used as a tool to examine the thermal and chemical denaturation of kB1. A 50 uM solution of kB1 in 10 mM potassium phosphate was examined by CD spectroscopy between 190 and 250 nm. The molar ellipticity at 220 nm was monitored with increasing temperature (5 C increments up to 90 C). No significant changes were observed in the CD spectra for either the pure kB1 solutions or solutions containing 8 M urea.
AssayType Chemical stability

References
Colgrave ML, Craik DJ (2004) Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43:5965-75

Cross-references
Proteins Assayed kalata B1