**2.3.5 Precast gels**

High-quality precast gels are preferred for high-throughput applications. They provide savings in time and labor, and the precision-poured gradients result in reproducibility among runs. Precast gels differ from handcast gels in that they are cast with a single buffer throughout and without SDS. During storage, different buffers in the stacking and resolving gels would mingle without elaborate means to keep them separate, and thus have no practical value. In addition, because the sample contains SDS, and the dodecyl sulfate ion in the cathode buffer moves faster than the proteins in the gel, keeping them saturated with the detergent, precast gels are made without SDS.

### **2.3.6 Transition from first to second dimension**

The transition from first-dimension to second-dimension gel electrophoresis involves two steps: equilibration of the resolved IPG strips in SDS reducing buffer, and embedding of the strip on the top of the second-dimension gel. Proper equilibration simultaneously ensures that proteins are coated with dodecyl sulfate and that cysteines are reduced and alkylated. The equilibrated IPG strips are placed on top of the gel and fixed with molten agarose solution to ensure good contact between the gel and the strip.

Two Dimensional Gel Electrophoresis in Cancer Proteomics 371

proteins and the residual background in the body of the gel (the signal-to-noise ratio). Unbound stain molecules can be washed out of the gels without removing much stain from the proteins. All stains interact differently with different proteins (Carroll *et al.* 2000). No stain will universally stain all proteins in a gel in proportion to their mass. The only observation that seems to hold for most stains is that they interact best with basic amino acids. For critical analysis, replicate gels should be stained with two or more different stains. Of all stains available, colloidal Coomassie Blue (Bio-Safe™ Coomassie) appears to stain the broadest spectrum of proteins. It is instructive, especially with 2-D PAGE gels, to stain a colloidal Coomassie Blue-stained gel with silver or to stain a fluorescently stained gel with colloidal Coomassie Blue or silver. Very often, this double staining procedure will show a few differences between the protein patterns. It is most common to stain gels first with Coomassie Blue or a fluorescent stain, and then restain with silver. However, the order in which the stains are used does not seem to be important, as long as the gels are washed well

Coomassie Brilliant Blue R-250 is the most common stain for protein detection in polyacrylamide gels. Coomassie Brilliant Blue R-250 and G-250 are wool dyes that have been adapted to stain proteins in gels. The "R" and "G" designations indicate red and green hues, respectively. Coomassie R-250 requires on the order of 40 ng of protein per spot for detection. Absolute sensitivity and staining linearity depend on the proteins being stained. The staining solution also fixes most proteins in gels. Bio-Safe Coomassie stain is made with Coomassie Brilliant Blue G-250. Bio-Safe Coomassie stain is a ready-touse, single-reagent protein stain. Sensitivity can be down to 10 ng, and greater contrast is achieved by washing the gel in water after staining. Used stain can be disposed of as

SYPRO Ruby protein gel stain has desirable features that make it popular in highthroughput laboratories. It is an endpoint stain with little background staining (high signalto noise characteristics) and it is sensitive and easy to use. SYPRO Ruby protein stain does not detect nucleic acids. SYPRO Ruby protein stain is sensitive to 1–10 ng and can be linear over 3 orders of magnitude. It is compatible with high through put protocols and downstream analysis, including mass spectrometry and Edman sequencing (Patton 2000). It also allows detection of glycoproteins, lipoproteins, low MW proteins, and metalloproteins that are not stained well by other stains. This fluorescent stain is easily visualized with simple UV or blue-light transilluminators, as well as by the Molecular Imager FX™ Pro Plus

Two popular methods for silver staining are recommended for 2-D analysis. They are based on slightly different chemistries but have similar sensitivities for protein. Bio-Rad's silver stain kit, based on the method of Merril *et al.* (1981), can be as much as 100 times more sensitive than Coomassie Blue R-250 dye staining and allows visualization of

nonhazardous waste and the procedure does not fix proteins in the gel.

with high-purity water between stains.

**2.4.3 SYPRO ruby fluorescent staining** 

multiimager and VersaDoc™ imaging systems.

**2.4.4 Silver staining** 

**2.4.2 Coomassie blue staining** 

### **2.3.7 Second dimension and high throughput**

Since the first dimension can be run in batches of 12–24 strips at a time, it is desirable to run the same number of samples in the second dimension. Precast gels ensure high reproducibility among samples and help reduce the work involved in running large numbers of samples. Alternatively, gels can be hand cast 12 at a time under identical conditions with multi-casting chambers. The Dodeca cells save time, space, and effort, and help to ensure that gels are run under the same electrical conditions for highest throughput and reproducibility.
