Detecting Cryptochrome Expression in Monarch Organs

To test if dpCRY1 was expressed in the putative magnetosensitive organs identified in behavioral experiments, antennae, compound eye photoreceptors, and optic lobes from five adult wild-type monarchs were dissected in Ringer’s solution and flash frozen. Proteins were extracted in 100 μl lysis buffer [150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 0.5% NP40, 1 mM EDTA, 1× Protease Inhibitor Tablet (Pierce)], and concentrations were measured using the Pierce Coomassie Plus Assay kit (Thermo Fisher). For each sample, 3 μg of protein was loaded per lane onto a 7.5% SDS-PAGE. DpCRY1 was detected using a guinea pig anti-dpCRY1 primary antibody (1:500; CRY1-GP37^{41 (link)}) and a peroxidase affinipure donkey anti-guinea pig IgG secondary antibody (1:1000; Jackson ImmunoResearch #706-035-148). Tubulin was detected using a mouse anti-α tubulin monoclonal antibody (1:10,000; Sigma B-5-1-2) and a goat anti-mouse IgG HRP secondary antibody (1:1000; Invitrogen, 31430).