Class III Peroxidases - Plant Proteomics: Methods and Protocols - page 658

destained, and digested by standard protocols for mass analysis [ 9 ,
41 ]. Membrane-bound peroxidases were identifi ed by matrix-
assisted laser desorption/ionization (MALDI) or electrospray ion-
ization (ESI) mass spectrometry in the past [ 9 , 18 ]. Identifi ed
peptides can be used for BLAST searches in any database available
in the World Wide Web. PeroxiBase, however, may be the pre-
ferred database for peroxidases. Although this chapter is focussed
on maize peroxidases, the protocols for PAGE can be applied to
other samples and enzyme activity stains too [ 40 ].

2 Materials

Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store all reagents at 4 °C (unless indicated
otherwise). All peroxidase activity staining solutions (50 mL) are
calculated for one mini-gel (ca. 10 cm × 7 cm).

2.1 Cell
Fractionation

A refrigerated centrifuge with angle rotors for 100 and 50 mL
tubes is used for preparation of microsomal fractions by differential
centrifugation. Plasma membranes and tonoplast are prepared with
a swing-out rotor for 50 mL tubes. Membrane fractions are col-
lected by ultracentrifugation with an angle rotor for 22 mL tubes
( see Note 1 ).

1. Homogenization buffer: 250 mM sucrose, 50 mM HEPES-
KOH, 1 mM EDTA, pH 7.5. Supplement buffer with 1 mM
DTT and 1 % polyvinylpyrrolidone (PVP) directly before use.
2. 100 mM phenylmethylsulfonyl fl uoride (PMSF) in 2-propanol.
3. Resuspension medium: 250 mM sucrose and 50 mM HEPES-
KOH, pH 7.0.

2.2 Soluble Fraction

1. Ammonium sulfate (Applichem, Germany).
2. Resuspension medium S (for soluble proteins): 50 mM Tris-
HCl and 1 mM EDTA, pH 7.5.

2.3 Plasma
Membrane Preparation
by Aqueous Polymer
Two-Phase
Partitioning

1. 20 % (w/w) Dextran solution. Weigh 100 g Dextran T500
powder (Pharma Cosmos, Denmark) and 400 g H 2 O. Transfer
fi rst the Dextran and then the H 2 O to a glass bottle and stir it
over night. Store the solution in freezer.
2. 40 % (w/w) Poly-ethylene glycol (PEG) solution: Weigh 100 g
PEG 3350 powder (Sigma-Aldrich, Germany) and 150 g H 2 O.
Transfer fi rst the PEG and then the H 2 O to a glass bottle and
solve it by stirring. Store the solution in freezer.

3. 200 mM PO 4 − buffer, pH 7.8: Prepare 100 mL Na 2 HPO 4
(2.84 g) and KH 2 PO 4 (2.72 g) solutions. Adjust Na 2 HPO 4
solution to pH 7.8 with the KH 2 PO 4 solution.
4. 200 mM KCl solution.