Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2

  • Małgorzata Szczygieł Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
  • Marcin Markiewicz Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
  • Milena Julia Szafraniec Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
  • Roxana Zuziak Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
  • Krystyna Urbańska Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
  • Leszek Fiedor Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
Keywords: Xenobiotic transporter, ABCG2, BCRP, Western blotting


All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP‑binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification.

The aim of this study was to optimize the methods for detection and isolation of BCRP‑enriched fractions obtained either from the cell cultures grown in vitro or animal tissue samples. In this report we describe an optimization of isolation of a BCRP‑enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.


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