Protein Protocols Protein Protocols

Disulfide Bridges 591
3. Spot the sample on the origin. This can be done using a micropipet fitted with a disposable
capillary tip. To keep the spot small, apply 0.5–1 µL spots and dry between
applications.
4. Apply 0.5 µL marker dye to the dye origin.
5. Set up electrophoresis apparatus with electrophoresis buffer in both buffer tanks. Prepare
electrophoresis wicks from Whatman 3MM paper, wet with buffer, and place in buffer tanks.
6. Prepare a blotter from a double sheet of Whatman 3MM, with holes cut at the positions of
the sample and marker origins. Wet with buffer and place over TLC plate. Ensure concentration
at the origins by pressing lightly around the holes.
7. Place TLC plate in apparatus.
8. Electrophorese at 1.5 kV for about 30–60 min.
9. Dry plate in a well-ventilated place at room temperature, overnight.
3.2. Performic Acid Oxidation (see Note 2)
1. Prepare performic acid by mixing 19 mL formic acid with 1 mL 30% (w/v) hydrogen
peroxide. The reaction is spontaneous. Care!
2. Place the dry electrophoresis sheet/plate in a container where it can be supported without
touching the sides.
3. Place the performic acid in a shallow dish inside the container. Close the container and
leave to oxidize for 2–3 h. (Note the marker dyes change from blue to green.)
4. Dry sheet thoroughly at room temperature overnight.
3.3. Second Dimension Electrophoresis
3.3.1. Paper Electrophoresis
1. To prepare for the second dimension, individual strips from the first dimension can be
machine zigzag stitched onto a second sheet. The overlap of the second sheet should
be carefully excised with a razor blade/scalpel.
2. Wet the sheet with electrophoresis buffer, applying the buffer along both sides of the
sample, thus concentrating the peptides in a straight line.
3. Repeat electrophoresis, at right angles to the original direction.
4. Thoroughly dry sheet, as before.
3.3.2. Thin-Layer Electrophoresis
1. Wet TLC plate with electrophoresis buffer using two sheets of prewetted Whatman 3 MM
paper on either side of sample line.
2. Repeat electrophoresis at right angles to the original direction.
3. Thoroughly dry plate as before.
3.4. Visualization
Peptide spots can be seen after reaction with fluorescamine.
1. The reaction should be carried out under alkaline conditions. The sheet or plate should be
dipped in a solution of triethylamine (1% [v/v]) in acetone. This should be carried out at
least twice if the electrophoresis buffer employed was acidic. Dry the sheet well.
2. Dip the sheet in a solution of fluorescamine in acetone (1 mg/100 mL).
3. Allow most of the acetone to evaporate.
4. View the map under a UV lamp at 300–365 nm (Care: Goggles must be worn). Peptides
and amino-containing compounds fluoresce. Encircle all fluorescent spots with
a soft pencil.