Biology Reference
In-Depth Information
1. Gel solution: 3 M urea, 2 % CHAPS, 10 % glycerol, 2 % carrier
ampholytes pH 3-10 (Serva, Germany), and 7.5 % AB-solution.
Solution is ultra sonicated until urea and CHAPS are solved.
2. Cathode buffer: 20 mM lysine and 20 mM arginine (Biorad,
Germany).
3. Anode buffer: 10 mM phosphoric acid.
4. 3× loading buffer (3× IEF-LB): 8 % Ampholyte, 8 % CHAPS,
40 % glycerol, 3 M urea.
2.11 Native IEF-PAGE
Components
2.12 High Resolution
Clear Native PAGE
(hrCNE) Components
1. 3× Gel buffer (3× hrCNE-GB): 75 mM imidazole/HCL and
1.5 M 6-aminocaproic acid (ACA), pH 7.0.
2. Cathode buffer: 50 mM Tricine, 7.5 mM imidazole, 0.05 %
deoxycholate, and 0.05 % triton X-100, pH 7.0. The pH is not
adjusted.
3. Anode buffer: 25 mM imidazole-HCl, pH 7.0.
4. Loading buffer (hrCNE-LB): 50 mM Imidazole-HCl, 500 mM
ACA, 1 mM EDTA, 20 % glycerol, and a trace of Ponceau S.
5. 20 % Digitonin (Sigma-Aldrich, Germany) dissolved in 50 %
glycerol by heating up to 95 °C.
Gel staining should be documented by a photo scanner or a digital
camera in Tag Image File Format (TIFF) and a resolution of 300-
600 DPI for further preparation.
2.13 Peroxidase
In-Gel Activity
Staining Components
1. 30 % H
2
O
2
stock solution (Sigma-Aldrich, Germany) stored at
4 °C.
2. TMB staining solution: Solve 22.5 mg TMB in 15 mL metha-
nol (
see
Note 4
). After the TMB is solved add 35 mL 250 mM
Na-acetate buffer pH 5.0.
3. DAB staining solution: 80 mg DAB (Sigma-Aldrich, Germany)
are solved in 1 mL dimethyl sulfoxide (DMSO), then add it to
50 mM Na-acetate buffer with 10 mM CaCl
2
, pH 5.0 and mix it.
4.
α
-Chloro-naphthol-staining solution: 0.01 %
α
-chloro-
naphthol in 0.1 M Na
2
HPO
4
buffer, pH 6.5.
5. Guaiacol-staining solution: 0.5 % (v/v) Guaiacol in 50 mM
Na-acetate buffer and 10 mM CaCl
2
, pH 5.0.
3
Methods
General cell fractionation is exemplifi ed for 5 days old maize roots
but can be applied similar to other maize root systems. For other
plants and tissues slight modifi cations might be needed for plasma
membrane and tonoplast preparation. All work steps are accom-
plished at 4 °C, if not mentioned otherwise.
3.1 Cell
Fractionation
