Expression of darcin in C57BL/6, BALB/c and wild male mice. Panel A: Urine from male adult mice of C57BL/6 (B6) or BALB/c (Bc) strains was resolved on SDS-PAGE at different loadings, from normal (2 μg of protein) to heavily overloaded (20 μg of protein) lanes. The high mobility band corresponding to darcin was evident at all loadings in C57BL/6 mice, but was barely evident at 10 μg and 20 μg loadings from BALB/c mice. Densitometric analysis of this gel indicated that darcin constituted approximately 15% of the total major urinary protein (MUP) in C57BL/6 mice, but less than 0.5% of total MUP in BALB/c mice. Panel B: Protein (5 μg) from a number of adult, male, wild-derived mice was analysed by SDS-PAGE. The darcin band, confirmed by mass spectrometry to be darcin, was evident in every animal, at levels similar to that seen in C57BL/6 mice. We do not have an explanation for the doublet in animal ID5343, but it is possible that this mouse is heterozygous for two darcin genes - the upper band has tryptic peptides that match to the darcin sequence .