Protein Protocols Protein Protocols

300 Jenö and Horst
and Coomassie Blue staining components from protein material. Based on a different
stationary phase, a similar approach was developed (7) to separate protein from
contaminants originating from the electroelution process. In this method, electroeluted
proteins are applied to a poly (2-hydroxyethyl) aspartamide-coated silica, which
provides a polar medium for binding of proteins when equilibrated at high organic
solvent concentration (8). Bound proteins are then eluted with a decreasing gradient of
organic modifier, allowing recovery of protein free of SDS and buffer salts. Although
the methodology is similar to the one described by Simpson et al. (6), proteins tend to
adsorb to the poly (hydroxyethyl) aspartamide matrix at lower n-propanol concentrations
than to reverse-phase matrices, therefore minimizing the danger of irreversible
protein precipitation on the stationary phase.
In this chapter, we describe an electroelution procedure that has worked well with
proteins in the molecular weight range of 20–100 kDa isolated from yeast mitochondrial
membranes. In addition, a method is described which allows the desalting of these
proteins into volatile buffer systems by hydrophilic-interaction chromatography.
2. Materials
1. Electrophoresis apparatus: Preparative electrophoresis is carried out on 16 × 10 cm separating
and 16 × 2 cm stacking gels of 1.5 mm thickness with the buffer system described
by Laemmli (9). For sample application, a preparative sample comb of 2 cm depth and
14 cm width is used. Up to 2.5 mg total protein is applied onto one preparative slab gel.
Samples are electrophoresed at 15 V for 14 h. Chemicals used for electrophoresis
(acrylamide, N,N'-methylene bis-acrylamide, ammonium persulfate, N,N,N',N'-tetramethylenediamine,
SDS, and Coomassie Blue R250) are electrophoresis-grade and are purchased
from Bio-Rad (Hercules, CA). Methanol and acetic acid used for staining are pro analysis
(p.a.) grade from Merck (Darmstadt, Germany). Unless stated, all other chemicals used
are of the highest grade available.
2. Staining solution: 0.125% (w/v) Coomassie Brilliant Blue R250, 50% (v/v) methanol,
10% acetic acid. Filter the staining solution over 320-µm filters (Schleicher and Schuell,
Dassel, Germany) before use.
3. Destaining solution: 50% methanol, 10% acetic acid.
4. Electroelution apparatus: BIOTRAP from Schleicher and Schuell (Dassel, Germany,
“Elutrap” trademark in the US and Canada).
5. BT1 and BT2 membranes for electroelution (Schleicher and Schuell).
6. Electroelution buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS. This buffer is prepared
with NANOpure water from a water purification system (Barnstead, Dubuque, IA, USA)
and electrophoresis-grade SDS (Bio-Rad).
7. Electrodialysis buffer: 15 mM NH4HCO3, 0.025% SDS. Prepare the buffer with
NANOpure water and electrophoresis-grade SDS (Bio-Rad).
8. High-pressure liquid chromatography (HPLC) equipment: we are using a Hewlett Packard
HP1090M (Palo Alto, CA) liquid chromatograph connected to a diode array UV detector
(Hewlett Packard, Palo Alto, CA) for operating columns of 4.6 × 200 mm or 2.1 mm
diameter.
9. Poly (hydroxyethyl) aspartamide (PHA) columns: 5 µm particle size, 20 nm pore size,
4. × 200 mm or 2.1 × 200 mm (PolyLC, Columbia, MD).
10. Solvents for hydrophilic interaction chromatography: solvent A: 100% n-propanol (HPLC
grade, Merck), 50 mM formic acid (Merck, analytical grade); solvent B: 50 mM formic
acid in water (NANOpure).