The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes

Erna Olivieri, Ben Herbert, Pier Giorgio Righetti*

*Corresponding author for this work

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The last few years have brought dramatic improvements for sample preparation and solubilization of protein for electrophoretic analyses. The use of reagents such as thiourea and novel sulfobetaine surfactants increases the total number of proteins able to be visualized from a complex mixture such as a cell lysate and also allows more hydrophobic membrane proteins to be resolved. As the red blood cell (RBC) contain no organelles, it is an ideal source of relatively pure plasma membrane for protein solubilization studies. In addition, there are a number of diseases related to abnormalities of RBCs proteins, thus it is of medical relevance as well as a test sample for technology development. However, the procedure for purifying RBC membranes is rather time-consuming and is normally carried out under almost physiological conditions, which can be conducive to proteolytic degradation of the membrane proteins. Significant differences in two-dimensional (2-D) patterns with and without protease inhibitors in sample preparation are demonstrated. In addition, is shown that preparation of RBC membranes with sodium carbonate, pH 11, leads to multimeric complexes of hemoglobin and causes hemoglobin to be irreversibly attached to the membrane. When using immobilized pH gradients (IPG) as the first dimension, it is demonstrated that the spectrins (large, filamentous proteins of 280 kDa) are lost from the 2-D map unless active, instead of passive, sample hydration into the IPG strip is adopted.

Original languageEnglish
Pages (from-to)560-565
Number of pages6
JournalElectrophoresis
Volume22
Issue number3
DOIs
Publication statusPublished - 2001

Keywords

  • Proteolysis
  • Red blood cell membranes
  • Spectrin
  • Two-dimensional maps

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