Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis

Electrophoresis
A VölklH D Fahimi

Abstract

Rat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen-specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197-208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich-technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well-preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolat...Continue Reading

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Citations

Aug 29, 2006·Analytical and Bioanalytical Chemistry·Ryan D JohnsonEdgar A Arriaga
Aug 8, 2007·Analytical and Bioanalytical Chemistry·Ralf J BraunMarius Ueffing
Apr 6, 1999·Trends in Biotechnology·W P Blackstock, M P Weir
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Nov 16, 2004·Proteomics·Angelika GörgMichael J Dunn
Jun 9, 2004·Biochemical and Biophysical Research Communications·Osamu KatsumataMiki Hara-Yokoyama
Aug 31, 2007·The Plant Journal : for Cell and Molecular Biology·Holger EubelA Harvey Millar
Sep 17, 2013·The FEBS Journal·Romain DrissiFrançois-Michel Boisvert
Oct 20, 2006·Biochimica Et Biophysica Acta·R A SaleemJ D Aitchison
Aug 17, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·A VölklH D Fahimi
Sep 18, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·H D Fahimi, E Baumgart
Jul 14, 1998·Electrophoresis·L Krivánková, P Bocek
May 28, 2010·Electrophoresis·Markus IslingerAlfred Völkl
Jan 30, 2008·Current Protocols in Cell Biology·Renate Fuchs, Isabella Ellinger
Nov 13, 2004·Proteomics·Joerg ReindersAlbert Sickmann

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