Protein Protocols Protein Protocols

290 Dunn
Fig. 1. A 2-DE separation of rat heart proteins visualized by SYPRO Ruby staining.
Note that the fluorescent image is shown as an inverted image to show black spots on a
white background for convenient viewing. The first dimension was pH 3–10 NL
immobilized pH gradient (IPG) 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. Method
Note: All incubations are carried out at room temperature with gentle agitation.
1. After electrophoresis, fix the gel immediately (see Note 2) in 500 mL (see Note 3) of
fixation solution for a minimum of 1 h at room temperature (see Note 4).
2. Place the gel into 500 mL of SYPRO Ruby protein gel stain solution for a minimum of 3 h
(see Note 5).
3. Wash the gel in 500 mL of washing solution for 10 min (see Note 6).
4. Image the gel (see Note 7).
4. Notes
1. SYPRO Ruby protein gel stain is available only as a premade solution from the manufacturers
(Molecular Probes, Eugene, OR, USA).