Yvon fluoromax 4 spectrofluorometer

Recombinant rat CaM was expressed in Escherichia coli (strain BL21) and purified using a phenyl-Sepharose affinity chromatography column as previously described [81 (link), 82 ]. The samples were dialyzed against ultrapure water and CaM was dansylated at room temperature with dansyl chloride as previously described [83 (link)]. Binding of the wild type and W/E-ΔK-S/E mutant peptides to CaM was determined adding increasing concentrations (0.1–12 μg/ml) of the peptides to 30 μg/ml dansyl-CaM in 1 ml of deionized water containing 10 mM CaCl₂ or 20 mM EGTA using a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer (Kyoto, Japan) exciting the sample at 340 nm and measuring the emission spectra at 450–600 nm.