human branching cholangiocyte organoids
Western blot showing JAG1 in healthy intrahepatic cholangiocytes (ICOs) and ICOs obtained from a patient suffering from Alagille Syndrome (AGS). AGS ICOs are heterozygous mutated in JAG1. Two large deletions were detected in mutated JAG1 (mJAG1) gene and protein. JAG1 and mJAG1 protein are both detected in AGS ICOs, only JAG1 is detected in healthy ICOs.
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Western Blot assay For western blotting, samples from healthy and AGS ICOs were obtained. The organoids were lysed with homemade 2x Laemmli Sample Buffer (161-0737) supplemented with 1,4-Dithithreitol (DTT; 0.1M) for 5 minutes at 95 °C. Protein concentrations were estimated with the bicinchoninic acid (BCA) protein assay (ThemoFisher Scientific, 23227) according to manufacturer’s protocol. Protein samples migrated through a sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (7%) in running buffer for 105 minutes at 100 V and were blotted to a polyvinlylideenfluoride membrane (Merck Chemicals BV, IPFL85R) using blotting buffer for 2 hours at 250 mA. Molecular weight of proteins was determined using precision plus protein dual color standard (Bio-Rad, 1610374) 250 kDa marker. To prevent nonspecific antibody binding, the membranes were blocked with Odyssey blocking buffer (Westburg BV, 927-40000) for 1 hour at room temperature. Subsequently, membranes were incubated with polyclonal antibody to JAG1 (anti-rabbit; 1:150, Biomatik, CAU25271) and monoclonal β-actin (anti-mouse; 1:1000, Santa Cruz Biotechnology, G3019) overnight at 4 °C. Followed by a 1 hour incubation with secondary antibodies (1:10000, IRDye 680LT (926-68020) and 800CW (926-32213), LI-COR Biosciences). Immunoblots were scanned by Odyssey 3.0 (Clx) imaging system and analyzed using Image Studio Lite (Version 5.2).