An optimized method for the isolation and identification of membrane proteins

Electrophoresis. 2003 Jun;24(11):1795-808. doi: 10.1002/elps.200305387.

Abstract

The purpose of this study was to develop a protocol suitable for membrane protein extraction from limited starting material and to identify appropriate conditions for two-dimensional (2-D) gel electrophoresis. We used A549 cells, a human alveolar type II cell line, and evaluated three protein extraction methods based on different separation principles, namely protein solubility, detergent-based and density-based organelle separation. Detergent-based extraction achieved the highest yield with 14.64% +/- 2.35 membrane proteins but sequential extraction with 7.35% +/- 0.78 yield and centrifugal extraction with 4.1% +/- 0.54 yield produced the purest fractionation of membrane proteins. Only the sequential and the detergent-based extraction proved suitable for small volumes of starting material. We identified annexin I + II, electron transfer flavoprotein beta-chain, H(+)-transporting ATP synthase, mitofilin and protein disulfide isomerase A3 as membrane and cytokeratin 8 + 18, actin and others as soluble proteins using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis and started to map the A549 cell proteome. Our data suggest that membrane proteins can be extracted efficiently from small samples using a simple sequential protein extraction method. They can be separated and identified successfully using optimized conditions in 2-D gel electrophoresis. The presented methods will be useful for further investigations of membrane proteins of alveolar and bronchial carcinomas.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation / methods
  • Cell Fractionation / standards
  • Cell Line
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Epithelial Cells / chemistry
  • Humans
  • Lung / cytology
  • Membrane Proteins / analysis
  • Membrane Proteins / isolation & purification*
  • Proteomics / methods*
  • Solubility
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Membrane Proteins