Technical noteDon't trust an(t)ybody - Pitfalls during investigation of candidate proteins for methylmercury transport at the placental interface
Introduction
The toxic organic mercury compound methylmercury crosses the placenta barrier most likely through active transport [1]. Our knowledge on the involved transporters is poor [2]. Mercury strongly adheres to sulfhydryl groups. Methylmercury-cysteine, a molecular mimicry of methionine [3], is transported into cells by L-type amino acid transporter (LAT) light chains LAT1/SLC7A5 and LAT2/SLC7A8 [4] dimerized to the 4F2 surface antigen heavy chain (4F2hc/CD98/SLC3A2). Exit from cells is assumed to occur as a complex with glutathione (GSH) recognized by GSH-carriers such as multidrug resistance-associated protein 2 (MRP2/ABCC2) [5].
In preparation to functional studies on placental mercury toxicokinetics, we aimed to verify specificity of commercial primary antibodies against LAT1, LAT2, 4F2hc, and MRP2 combining the methods of immunoblotting [6] with target-specific small interfering RNA (siRNA)-mediated knockdown [7]. Current literature suggests that antibody specificity is valid only in 50% of cases [8]. Many researchers worldwide, who invested incredible amounts of money and time into validation, realized this [9], [10], [11]. Despite our awareness that antibodies do not necessarily recognize (only) their specified target, we were quite surprised by the results obtained during validation and want to share the most striking observations with the research community.
Section snippets
Materials and methods
We selected one antibody against 4F2hc, two antibodies against LAT1 and LAT2, respectively, and one further against MRP2 according to two criteria, i.e., recommendation for immunoblotting and provision of immunoblot(s) indicating specificity for a protein of the appropriate size.
Healthy term placentas were obtained after elective cesarean sections. Prior to, all patients gave written informed consent and the study was approved by the ethics committee of the Medical University Vienna (EC-number
System L
The Aviva 4F2hc antibody detected a single placental protein of around 80–90 kDa (Fig. 1A), which is in agreement with previous studies [14]. In BeWo and HeLa cells we detected two bands (Fig. 1A) likely representing two differently glycosylated proteins [15], [16]. Both LAT1 antibodies (Abcam, Fig. 1B; Epitomics, Fig. 1C) detected an appropriate major protein at 40–50 kDa. The LAT2 antibodies (Santa Cruz, Fig. 1D; Abcam, Fig. 1E) showed strong cross-reactivity with albumin in placental lysates
Acknowledgements
The study was supported by LifeScience2010 (Project LS10026), NFB (Niederösterreichische Forschungs- und Bildungsgesellschaft). We thank Prof. John Robinson for providing the highly enriched apical plasma membrane fractions of placental trophoblasts.
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Faculty of Medicine, Mahasarakham University, Thailand.