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

Isabella Ellinger; Waranya Chatuphonprasert; Martin Reiter; Agnes Voss; Jost Kemper; Elisabeth Straka; Matthias Scheinast; Harald Zeisler; Hans Salzer; Claudia Gundacker

doi:10.1016/j.placenta.2016.04.011

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

Placenta. 2016 Jul:43:13-6. doi: 10.1016/j.placenta.2016.04.011. Epub 2016 Apr 20.

Authors

Affiliations

¹ Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: [email protected].
² Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: [email protected].
³ Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: [email protected].
⁴ Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: [email protected].
⁵ Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address: [email protected].
⁶ Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: [email protected].
⁷ Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: [email protected].
⁸ Department of Obstetrics and Gynecology, Medical University Vienna, Austria. Electronic address: [email protected].
⁹ Clinic for Pediatrics and Adolescent Medicine, Landesklinikum Tulln, Tulln, Austria. Electronic address: [email protected].
¹⁰ Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University Vienna, Vienna, Austria. Electronic address: [email protected].

PMID: 27324094
DOI: 10.1016/j.placenta.2016.04.011

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.

Keywords: ABCC2 protein; Antibody validation; Human placenta; SLC3A2 protein; SLC7A5 protein; SLC7A8 protein.

MeSH terms

Adaptor Proteins, Signal Transducing / genetics
Adaptor Proteins, Signal Transducing / metabolism
Biological Transport
Carrier Proteins / metabolism*
Female
Fusion Regulatory Protein 1, Heavy Chain / genetics
Fusion Regulatory Protein 1, Heavy Chain / metabolism
Humans
Large Neutral Amino Acid-Transporter 1 / genetics
Large Neutral Amino Acid-Transporter 1 / metabolism
Methylmercury Compounds / metabolism*
Multidrug Resistance-Associated Protein 2
Multidrug Resistance-Associated Proteins / genetics
Multidrug Resistance-Associated Proteins / metabolism
Placenta / metabolism*
Pregnancy
RNA, Small Interfering

Substances

ABCC2 protein, human
Adaptor Proteins, Signal Transducing
Carrier Proteins
Fusion Regulatory Protein 1, Heavy Chain
LAT2 protein, human
Large Neutral Amino Acid-Transporter 1
Methylmercury Compounds
Multidrug Resistance-Associated Protein 2
Multidrug Resistance-Associated Proteins
RNA, Small Interfering