Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 79
Approximately 20–30 µg of protein is transferred to a 1.5-mL tube and mixed
(1:5) with 6X loading buffer containing 10% β-mercaptoethanol. Vortex sample
shortly (5 s) and boil for 5–8 min to denature the proteins before loading on 1D gel.
For most purposes, a 10% gel is recommended, but alternatively a gradient gel
can be used (4–12%) for higher resolution in a broad molecular mass range.
The parameter for running the gel depends on the gel apparatus system and gel
size and has to be done according to the individual manufacturer’s instructions.
3.3.4. Staining of Proteins After 1D Gel Electrophoresis
After resolving the proteins by 1D gel electrophoresis, the proteins are visualized
by either silver staining (49) or colloidal Coomassie (50). Silver staining
can sensitize proteins down to 1–2 ng of protein (51), whereas colloidal
Coomassie staining can detect levels down to approx 10–20 ng of protein. Both
methods are equally compatible with mass spectrometry analysis.
3.3.5. Visualization of Proteins by Silver Staining
1. After resolving the proteins by 1D gel electrophoresis, fix the gel in destaining
solution (50% methanol, 5% acetic acid, 45% water [Milli-Q], [v/v]) for 20–30 min
at room temperature (on shaker).
2. Rinse in Milli-Q water for 30–60 min to remove acid (change water three to four
times). However, the gel can be left overnight in water to reduce background
(more transparent in areas with no protein staining).
3. Incubate the gel for 1 to 2 min in 0.02% sodium thiosulfate (Na2S2O3 ).
4. Wash two times with Milli-Q water (1 min each time).
5. Then, incubate the gel in ice-cold (4°C) 0.1% silver-nitrate (AgNO3 ) solution at
4°C for 20–40 min (on shaker).
6. Subsequently, wash the gel two times in Milli-Q water (two times for 1 min each)
and then develop in developing solution (0.04% formaldehyde/formalin and 2%
Na2CO3 ). The development is quenched by discharging the developing solution
and replacing it with 1% acetic acid (see Note 24). After staining (silver or colloidal
Coomassie), the gel is stored at 4°C in 1% acetic acid (container with tight
lid). The gel can be stored for several months under these conditions.
3.3.6. Visualization of Proteins by Colloidal Coomassie Staining
1. After gel electrophoresis, fix the gel in destaining solution (50% methanol,
5% acetic acid, and 45% water [Milli-Q] [v/v]) for 10 min at room temperature
(on shaker).
2. Discharge the destaining solution and replace it by staining solution without
stainer B (55 mL of Milli-Q water, 20 mL of methanol, 20 mL of stainer A).
3. Incubate at room temperature for 10 min (on shaker).
4. Add 5 mL of stainer B to the existing solution and leave for 3–12 h.
5. When protein bands become visible, decant staining solution and replace by
200–300 mL of Milli-Q water.