title: Quantitative immunofluorescent blotting of the multidrug resistance-associated protein 2 (MRP2).
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.affiliation: 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
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Gerk
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002138:Calibration; D002460:Cell Line; D006801:Humans; D007202:Indicators and Reagents; D057230:Limit of Detection; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D007202:Indicators and Reagents; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C000715087:ABCC2 protein, human