Don't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface.
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.
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: isabella.ellinger@meduniwien.ac.at.
Isabella
I
Ellinger
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: waranya.c@msu.ac.th.
Waranya
W
Chatuphonprasert
Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: martinreiter@yahoo.de.
Martin
M
Reiter
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: n0942129@students.meduniwien.ac.at.
Agnes
A
Voss
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: n0842190@students.meduniwien.ac.at.
Jost
J
Kemper
Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: elisabeth.straka@meduniwien.ac.at.
Elisabeth
E
Straka
Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: matthias.scheinast@meduniwien.ac.at.
Matthias
M
Scheinast
Department of Obstetrics and Gynecology, Medical University Vienna, Austria. Electronic address: harald.zeisler@meduniwien.ac.at.
Harald
H
Zeisler
Clinic for Pediatrics and Adolescent Medicine, Landesklinikum Tulln, Tulln, Austria. Electronic address: hans.salzer@meduniwien.ac.at.
Hans
H
Salzer
Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: claudia.gundacker@meduniwien.ac.at.
Claudia
C
Gundacker
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D005260:Female; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000235:genetics / Q000378:metabolism; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism; D008767:Methylmercury Compounds / Q000378:metabolism*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D034741:RNA, Small Interfering
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; D008767:Methylmercury Compounds; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D034741:RNA, Small Interfering; C000715087:ABCC2 protein, human; C419196:LAT2 protein, human
ABCC2 protein; Antibody validation; Human placenta; SLC3A2 protein; SLC7A5 protein; SLC7A8 protein