Protein Protocols Protein Protocols

268 Dunn
Fig. 2. Two-DE Separation of human heart proteins visualized by silver staining. A loading
of 100 µg protein was used. The first dimension was pH 3–10 NL immobilized pH gradient
(IPG) isoelectric focusing (IEF) and the second dimension was 12% T SDS-PAGE. The scale
at the top indicates the nonlinear pH gradient used in the first IPG 3–10 NL IEF dimension,
while the scale at the left indicates M r × 10 –3 .
3.1. Fixation
1. After electrophoresis, fix the gel immediately (see Note 1) in 200 mL (see Note 2) of TCA
(see Note 3) for a minimum of 1 h at room temperature. High-percentage polyacrylamide
and thick gels require an increased period for fixation, and overnight soaking is recommended.
2. Place the gel in 200 mL of 40% (v/v) ethanol, 10% (v/v) acetic acid in water and soak for
2 × 30 min (see Note 4).
3. Wash the gel in excess water for 2 × 20 min, facilitating the rehydration of the gel and the
removal of methanol. An indication of rehydration is the loss of the hydrophobic nature of
the gel.
3.2. Sensitization
1. Soak the gel in a 10% (w/v) glutaraldehyde solution for 30 min at room temperature (see
Note 5).
2. Wash the gel in water for 3 × 20 min to remove excess glutaraldehyde.