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Class III Peroxidases

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

Abstract

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 

Abbreviations

2D

Two dimensional

AB

Acrylamide/Bis

ACA

Aminocaproic acid, 6-aminohexanoic acid

APS

Ammonium persulfate

BCA

Bicinchoninic acid

BLAST

Basic Local Alignment Search Tool

BN-PAGE

Blue native PAGE

BSA

Bovine serum albumin

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

DAB

3′3′-Diaminobenzidine

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

EDTA

Ethylenediaminetetraacetic acid

ESI

Electrospray ionization

GB

Gel buffer

Guaiacol

2-Methoxyphenol

HEPES

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

hrCNE

High resolution clear native electrophoresis

ICM

Intracellular membranes

IEF

Isoelectric focussing electrophoresis

LB

Loading buffer

MALDI

Matrix-assisted laser desorption/ionization

modSDS-PAGE

Modified SDS-PAGE

PAGE

Polyacrylamide gel electrophoresis

PEG

Poly-ethylene glycol

PM

Plasma membrane

PMSF

Phenylmethylsulfonyl fluoride

PVPP

Polyvinylpolypyrrolidone

TEMED

N,N,N,N′-Tetramethyl-ethylenediamine

TIFF

Tag Image File Format

TMB

Tetramethylbenzidine

Tris

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

Notes

Acknowledgments

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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