Protein Protocols Protein Protocols

590 Aitken and Learmonth
3. Whatman (Maidstone, UK) 3MM and Whatman No. 1 paper.
4. Cellulose TLC plates (Machery Camlab [Cambridge, UK] Nagel or Merck [Poole, UK]).
2.2. Reagents
1. Electrophoresis buffers (see Note 1): Commonly used volatile buffers are:
pH 2.1 acetic acid/formic acid/water 15/5/80, v/v/v
pH 3.5 pyridine/acetic acid/water 5/50/945 v/v/v
pH 6.5 pyridine/acetic acid/water 25/1/225, v/v/v
2. Nonmiscible organic solvents: toluene, for use with pH 6.5 buffer: white spirit for use
with pH 2.1 and 3.5 buffers.
3. Formic acid. Care!
4. 30% w/v Hydrogen peroxide. Care!
5. Fluorescamine: 1 mg/100 mL in acetone.
6. Marker dyes: 2% orange G, 1% acid fuschin, and 1% xylene cyanol dissolved in appropriate
electrophoresis buffer.
7. 1% (v/v) Triethylamine in acetone.
3. Methods
3.1. First-Dimension Electrophoresis
3.1.1. Paper Electrophoresis
1. Dissolve the peptide digest from 0.1–0.3 µmol protein in 20–25 µL electrophoresis buffer.
2. Place the electrophoresis sheet on a clean glass sheet (use Whatman No. 1 for analytical
work, Whatman 3MM for preparative work). Support the origin at which the sample is to
be applied on glass rods. Where paper is used, multiple samples can be run side by side.
Individual strips can then be cut out for running in the second dimension.
3. Apply the sample slowly without allowing to dry (covering an area of about 2 × 1 cm)
perpendicular to the direction intended for electrophoresis. NB: for pH 6.5 buffer, apply
near the center of the sheet, for acidic buffers, apply nearer the anode end.
4. Apply a small volume of marker dyes (2% orange G, 1% acid fuschin, 1% xylene cyanol,
in electrophoresis buffer) on the origin and additionally in a position that will not overlap
the peptides after the second dimension.
5. Once the sample is applied, wet the sheet with electrophoresis buffer slowly and uniformly
on either side of the origin so that the sample concentrates in a thin line. Remove
excess buffer from the rest of the sheet with blotting paper.
6. Place the wet sheet in the electrophoresis tank previously set up with electrophoresis buffer
covering bottom electrode. An immiscible organic solvent (toluene where pH 6.5 buffer is
used, white spirit for acidic buffers) used to fill the tank to the top. Upper trough filled
with electrophoresis buffer. Care!
7. Electrophorese at 3 kV for about 1 h, with cooling if necessary. The progress of the electrophoresis
can be monitored by the movement of the marker dyes (see Note 1).
8. Dry sheet in a well-ventilated place overnight, at room temperature, secured from a glass
rod with Bulldog clips.
3.1.2. Thin-Layer Electrophoresis
1. Dissolve the peptide digest from 0.1–0.3 µmol protein in at least 10 µL electrophoresis
buffer.
2. Mark the sample and dye origins on the cellulose side of a TLC plate with a cross using
an extrasoft blunt-ended pencil, or on the reverse side with a permanent marker.