Phillip M Gerk1. 1. Dept. of Pharmaceutics, Virginia Commonwealth University, MCV Campus, School of Pharmacy, Room 454F Smith Bldg, 410 N. 12th Street, P.O. Box 980533, Richmond, VA 23298–0533, USA. pmgerk@vcu.edu
Abstract
INTRODUCTION: Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. PURPOSE: The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-binding cassette transporter isoform C2/multidrug resistance-associated protein 2 (ABCC2/MRP2). METHODS: Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. RESULTS: The limits of quantitation for the time-insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. DISCUSSION: The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.
INTRODUCTION: Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. PURPOSE: The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-binding cassette transporter isoform C2/multidrug resistance-associated protein 2 (ABCC2/MRP2). METHODS: Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. RESULTS: The limits of quantitation for the time-insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. DISCUSSION: The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.
Authors: F Azzaroli; A Mennone; V Feletti; P Simoni; E Baglivo; M Montagnani; N Rizzo; G Pelusi; D DE Aloysio; F Lodato; D Festi; A Colecchia; E Roda; J L Boyer; G Mazzella Journal: Aliment Pharmacol Ther Date: 2007-10-15 Impact factor: 8.171