Class III Peroxidases

  • Sabine Lüthje
  • Claudia-Nicole Meisrimler
  • David Hopff
  • Tim Schütze
  • Jenny Köppe
  • Katrin Heino
Part of the Methods in Molecular Biology book series (MIMB, volume 1072)


Class III peroxidases are heme-containing proteins of the secretory pathway with an extremely high number of isoenzymes, indicating the tremendous and important functions of this protein family. This chapter describes fractionation of the cell in subproteomes, their separation by polyacrylamide gel electrophoresis (PAGE) and visualization of peroxidase isoenzymes by heme and specific in-gel staining procedures. Soluble and membrane-bound peroxidases were separated by differential centrifugation. Aqueous polymer two-phase partitioning and discontinuous sucrose density gradient were applied to resolve peroxidase profiles of plasma membranes and tonoplast. Peroxidase isoenzymes of subproteomes were further separated by PAGE techniques such as native isoelectric focussing (IEF), high resolution clear native electrophoresis (hrCNE), and modified sodium dodecyl sulfate (modSDS)-PAGE. These techniques were used as stand-alone method or in combination for two-dimensional PAGE.

Key words

Cell fractionation Plasma membrane Tonoplast hrCNE IEF modSDS-PAGE Peroxidase in-gel staining 



Two dimensional




Aminocaproic acid, 6-aminohexanoic acid


Ammonium persulfate


Bicinchoninic acid


Basic Local Alignment Search Tool


Blue native PAGE


Bovine serum albumin






Dimethyl sulfoxide




Ethylenediaminetetraacetic acid


Electrospray ionization


Gel buffer




4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


High resolution clear native electrophoresis


Intracellular membranes


Isoelectric focussing electrophoresis


Loading buffer


Matrix-assisted laser desorption/ionization


Modified SDS-PAGE


Polyacrylamide gel electrophoresis


Poly-ethylene glycol


Plasma membrane


Phenylmethylsulfonyl fluoride






Tag Image File Format




Tris(hydroxymethyl)aminomethane 2-amino-2-hydroxymethyl-propane-1,3-diol



This work was supported by the Deutsche Forschungsgemeinschaft (DFG Lu 668/4-4) and the University of Hamburg (Young Researcher Initiative grant to C.N. Meisrimler and PhD student grant to D. Hopff).


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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Sabine Lüthje
    • 1
  • Claudia-Nicole Meisrimler
    • 2
  • David Hopff
    • 1
  • Tim Schütze
    • 1
  • Jenny Köppe
    • 1
  • Katrin Heino
    • 1
  1. 1.Biocentre Klein Flottbek, University of HamburgHamburgGermany
  2. 2.Leibniz Institute of Plant Genetics and Crop Plant ResearchGaterslebenGermany

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