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

• 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.

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.