2.3. Western Blotting
For this technique, the equipment only allowed for 8 samples to be tested at once. For the month of cull study, we simply compared 4 animals from January and 4 animals from November. For the animal feeding behaviour study, we compared 3 male adult animals from the consistent beggar and rare beggar groups, plus one male juvenile consistent beggar and one female consistent beggar. This was important to do because our previous studies had shown that UT-B2 abundance was comparatively low in male adults (Zhong et al., 2022c). We therefore wanted an indication if this was still the same in consistent beggars. Protein samples were prepared using ~25cm2 sections of rumen tissue for each sample. Tissues were thoroughly washed in 1X PBS solution and then homogenized in 5 ml of homogenisation buffer (300 mM mannitol, 12 mM HEPES, pH 7.6), using a polytron homogeniser (Kinematica, Switzerland). The resulting homogenates were spun at 1,000g at 4oC for 5 min, before the pellet was discarded, and the remaining supernatant was span at 17,000g at 4oC for 30 min. This second resulting pellet contained plasma membrane-enriched protein and was resuspended in homogenisation buffer, whilst the supernatant liquid contained cytosolic-enriched protein and was not utilised in this study. Prior to sample loading, 4X reducing Laemmli sample buffer (5% SDS, 25% glycerol, 0.32 M Tris, pH 6.8, bromophenol blue; Biorad, USA) was added to the protein samples and they were heated at 70oC for 10 min. Samples were ran on 8-16% polyacrylamide gels (Biorad, USA) for 60 minutes at 160V, loading ~10 µg protein per lane. Proteins were transferred to nitrocellulose membranes and probed for 16 hours at room temperature in 1:1000 UT-Bc19 or 1:2000 anti-MCT1 antibodies. Membranes were washed and probed with 1:5,000 horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-chicken respectively) for 1 hour at room temperature. Blots were further washed, then detection of protein performed using ECL reagents (PerkinElmer, UK) and a LAS-4000 Image Reader (Fujifilm, Japan). Lastly, densitometry analysis of images was performed using ImageJ software (National Institute of Health, USA).