Don't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface. (July 2016)

• Record Type: Journal Article
• Authors: Ellinger, Isabella; Chatuphonprasert, Waranya; Reiter, Martin; Voss, Agnes; Kemper, Jost; Straka, Elisabeth; Scheinast, Matthias; Zeisler, Harald; Salzer, Hans; Gundacker, Claudia
• Is Part Of: Placenta. Volume 43(2016:Jul.)
• Page Start: 13
• Page End: 16
• Publication Date: 2016-07
• Subjects: Antibody validation -- SLC3A2 protein -- SLC7A5 protein -- SLC7A8 protein -- ABCC2 protein -- Human placenta; Placenta -- Periodicals; Reproduction -- Periodicals; Placenta -- Periodicals; Placenta -- Périodiques; Reproduction -- Périodiques; 612.63
• DOI: 10.1016/j.placenta.2016.04.011
• Languages: English
• ISSNs: 0143-4004

Abstract

Abstract: While investigating placental mercury transport, we validated specificity of commercial antibodies against four candidate transporters (Large neutral amino acids transporter (LAT)1, LAT2, 4F2 cell-surface antigen heavy chain (4F2hc), and multidrug resistance-associated protein (MRP)2) by immunoblotting and small interfering RNA (siRNA)-mediated protein knockdown. An anti-4F2hc- and one anti-LAT1-antibody were specific. Another anti-LAT1-antibody reacted with LAT2. Two anti-LAT2-antibodies detected mainly albumin in placental lysates. A specific anti-MRP2-antibody hardly detected MRP2 in human placentas, contradicting published data. We recommend testing any unknown antibody by western blotting for 1/specificity for the protein of interest using e.g . siRNA knockdown and 2/cross-reactivity with albumin. Highlights: Specificity of commercial antibodies against LAT1, LAT2, 4F2hc, MRP2 was validated. We found an anti-LAT1 antibody to actually react with LAT2 instead of LAT1. Two anti-LAT2 antibodies detected mainly albumin in placental lysates. A specific antibody hardly detected MRP2 in human placentas, contradicting published data. Our findings underpin the need for rigorous validation of commercial antibodies.