Two‐dimensional electrophoresis of membrane and cytosol proteins of mouse liver and brain

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Abstract
Membrane proteins and cytosol proteins from organs (liver, brain) of inbred mice were separated by two‐dimensional electrophoresis (2DE). The 2DE technique revealed high resolution of complex protein solutions by staining the proteins. The 2DE patterns of membrane proteins showed about 660 protein spots (polypeptides) for both liver and brain. The protein patterns of the cytosols revealed about 800 polypeptides for the liver and about 680 for the brain. The membrane proteins and cytosol proteins of the 2DE patterns represented two protein populations specific for these two cell components. They matched in 7 % (liver) or 23 % (brain) of the total number of membrane protein spots. The matched spots represented dissociable membrane proteins rather than contamination of the isolated membrane fractions by cytosol proteins. Protein patterns of the same cell fraction but from the two different organs investigated contained more than 50 % organ‐unspecific polypeptides.Several conditions of our 2DE standard procedure were reinvestigated to test the usefulness for our technique of some methodical steps employed in 2DE by other authors. We found that many steps often used in sample preparations impaired rather than improved the 2DE pattern. In particular, addition of SDS to the protein sample resulted in a considerable loss of proteins during isoelectric foucing. The 2DE itself could be simplified by omitting several steps such as prefocusing, equilibration of focusing gels, forming gel gradients, autoradiography. This has some implications for the development of 2DE to a routine method.