Goat anti rabbit or rabbit anti goat igg h l hrp conjugate

Protein extraction and quantification, and Western blot protocol were performed as previously described^{48 (link)}. Briefly, the samples (containing 20 µg protein) were boiled, electrophoresed and transferred to a nitrocellulose membrane (Bio-Rad). After blocking using 1X RapidBlock solution (AMRESCO, Toronto, ON, Canada), target proteins within the membrane were detected by overnight incubation with specific primary antibody: goat polyclonal to GLUT2 (1:500 dilution; Catalog # ab111117; Abcam) and rabbit polyclonal to SGLT1 (1:500 dilution; Catalog # ab14686; Abcam). Vinculin protein was used for normalization and was detected using rabbit antiserum directed against mouse vinculin (1:1000 dilution; Catalog # ab129002, Abcam). As secondary antibodies, goat anti-rabbit or rabbit anti-goat IgG (H + L) HRP conjugate (Bio-Rad) diluted 1:2000 were used. Protein were visualized using Clarity™ Western ECL substrate (Bio-Rad) in a ChemiDoc™ MP imaging system (Bio-Rad) with chemiluminescence detection. Blot images were analysed using ImageLab software and band density of vinculin was used to normalize glucose transporter protein density.

For Western blot analysis we chose the concentrations and time in which ghrelin exerts the most significant inductions in mRNA expression. Protein extraction and quantification, and Western blot protocol were performed as previously described68 (link). Briefly, the samples (containing 30 μg protein) were boiled, electrophoresed and transferred to a nitrocellulose membrane (Bio-Rad). After blocking, target proteins within the membrane were detected by overnight incubation in 1x RapidBlock™ solution containing specific primary antibody (see Immunohistochemistry section for information on antibodies used). Vinculin protein was used for normalization and was detected using rabbit antiserum directed against mouse vinculin (1:2000 dilution; Catalog ≠ ab129002, Abcam). As secondary antibodies, goat anti-rabbit or rabbit anti-goat IgG (H + L) HRP conjugate (Bio-Rad) diluted 1:3000 were used. Protein were visualized using Clarity™ Western ECL substrate (Bio-Rad) in a ChemiDoc™ MP imaging system (Bio-Rad) with chemiluminescence detection. Blot images were plotted using ImageJ software and band density of vinculin was used to normalize glucose transporters protein density.