**2.2.10 Power conditions and resolution in IEF**

During an IEF run, the electrical conductivity of the gel changes with time, especially during the early phase. When an electrical field is applied to an IPG at the beginning of an IEF run, the current will be relatively high because of the large number of charge carriers present.

Two Dimensional Gel Electrophoresis in Cancer Proteomics 369

they allow proteins with a wide range of MW to be analyzed simultaneously, and the

The percentage of acrylamide, often referred to as %T (total percentage of acrylamide plus crosslinker) determines the pore size of a gel. Most protein separations use 37.5 parts acrylamide to 1 part bis-acrylamide (bis). Some researchers substitute piperazine bisacrylamide (PDA), which can reduce silver staining background and give higher gel strength. If the total percentage of acrylamide plus crosslinker is higher, the smaller is the pore size. A suitable %T can be estimated from charts of mobility for proteins of

Gradient gels are cast with acrylamide concentrations that increase from top to bottom so that the pore size decreases as proteins migrate further into the gels. As proteins move through gradient gels from regions of relatively large pores to regions of relatively small pores, their migration rates slow. Small proteins remain in gradient gels much longer than they do in single-percentage gels that have the same average %T, so both large and small molecules may be resolved in the same gel. This makes gradient gels popular for analysis of complex mixtures that span wide MW ranges. A gradient gel, however, cannot match the resolution obtainable with a properly chosen single concentration of acrylamide. A good approach is to use gradient gels for estimates of the complexities of mixtures. A proteomics experiment might start out with an 8–16%T gradient for global comparison. After interesting regions of the 2-D array have been identified, a new set of single-percentage gels may be run to study a particular size range of proteins. It is simplest and often most cost and labor

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

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

decreasing pore size along the gradient functions to sharpen the spots.

effective to purchase commercially available precast gradient gels.

detergent, precast gels are made without SDS.

**2.3.6 Transition from first to second dimension** 

solution to ensure good contact between the gel and the strip.

**2.3.3 Single-percentage gels** 

different MW.

**2.3.4 Gradient gels** 

**2.3.5 Precast gels** 

As the proteins and ampholytes move toward their pIs, the current will gradually decrease due to the decrease in the charge on individual proteins and carrier ampholytes. The pH gradient, strip length, and the applied electrical field determine the resolution of an IEF run. According to both theory and experiment, the difference in pI between two adjacent IEFresolved protein bands is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient at the position of the bands (Garfin 2000). Thus, narrow pH ranges and high-applied voltages yield high resolution in IEF. The highest resolution can be achieved using micro-range IPG strips and an electrophoretic cell, such as the PROTEAN IEF cell, capable of applying high voltages. IEF runs should always be carried out at the highest voltage compatible with the IPG strips and electrophoretic cell. However, high voltages in electrophoresis are accompanied by large amounts of generated heat. The magnitude of the electric field that can be applied and the ionic strength of the solutions that can be used in IEF are limited. Thin gels are better able to dissipate heat than thick ones and are therefore capable of withstanding the high voltage that leads to higher resolution. Also, at the completion of focusing, the current drops to nearly zero since the carriers of the current have stopped moving. The PROTEAN IEF cell is designed to provide precise cooling, allowing the highest possible voltages to be applied. (A default current limit of 50 μA per strip is intended to minimize protein carbamylation reactions in urea sample buffers. This limit can be increased to 99 μA per strip.)

Consistent and reproducible focusing requires that the time integral of voltage (volt-hours) be kept consistent. It is usually necessary to program IEF runs to reach final focusing voltages in stages. This approach clears ionic constituents in the sample from the IPG strips while limiting electrical heating of the strips. The PROTEAN IEF cell allows for multistep runs at durations set by time or volt-hours. The number of volt-hours required to complete a run must be determined empirically. A more complex sample in terms of number of proteins or even a different sample buffer might require increased volt-hours. The time needed to achieve the programmed volt-hours depends on the pH range of the IPG strip used as well as sample and buffer characteristics. If different strips are run at the same time, the electrical conditions experienced by individual strips will be different, perhaps exposing some strips to more current than desired, since the total current limit is averaged over all strips in a tray.

#### **2.3 The second dimension: SDS-PAGE**

#### **2.3.1 Protein separation by molecular weight (MW)**

Second-dimension separation is by protein mass, or MW, using SDS-PAGE. The proteins resolved in IPG strips in the first dimension are applied to second-dimension gels and separated by MW perpendicularly to the first dimension. The pores of the second-dimension gel sieve proteins according to size because dodecyl sulfate coats all proteins essentially in proportion to their mass. The net effect is that proteins migrate as ellipsoids with a uniform negative chargeto- mass ratio, with mobility related logarithmically to mass (Garfin 1995).

#### **2.3.2 Gel composition**

Homogeneous (single-percentage acrylamide) gels generally give excellent resolution of sample proteins that fall within a narrow MW range. Gradient gels have two advantages: they allow proteins with a wide range of MW to be analyzed simultaneously, and the decreasing pore size along the gradient functions to sharpen the spots.

#### **2.3.3 Single-percentage gels**

368 Gel Electrophoresis – Advanced Techniques

As the proteins and ampholytes move toward their pIs, the current will gradually decrease due to the decrease in the charge on individual proteins and carrier ampholytes. The pH gradient, strip length, and the applied electrical field determine the resolution of an IEF run. According to both theory and experiment, the difference in pI between two adjacent IEFresolved protein bands is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient at the position of the bands (Garfin 2000). Thus, narrow pH ranges and high-applied voltages yield high resolution in IEF. The highest resolution can be achieved using micro-range IPG strips and an electrophoretic cell, such as the PROTEAN IEF cell, capable of applying high voltages. IEF runs should always be carried out at the highest voltage compatible with the IPG strips and electrophoretic cell. However, high voltages in electrophoresis are accompanied by large amounts of generated heat. The magnitude of the electric field that can be applied and the ionic strength of the solutions that can be used in IEF are limited. Thin gels are better able to dissipate heat than thick ones and are therefore capable of withstanding the high voltage that leads to higher resolution. Also, at the completion of focusing, the current drops to nearly zero since the carriers of the current have stopped moving. The PROTEAN IEF cell is designed to provide precise cooling, allowing the highest possible voltages to be applied. (A default current limit of 50 μA per strip is intended to minimize protein carbamylation

reactions in urea sample buffers. This limit can be increased to 99 μA per strip.)

strips in a tray.

**2.3.2 Gel composition** 

**2.3 The second dimension: SDS-PAGE** 

**2.3.1 Protein separation by molecular weight (MW)** 

Consistent and reproducible focusing requires that the time integral of voltage (volt-hours) be kept consistent. It is usually necessary to program IEF runs to reach final focusing voltages in stages. This approach clears ionic constituents in the sample from the IPG strips while limiting electrical heating of the strips. The PROTEAN IEF cell allows for multistep runs at durations set by time or volt-hours. The number of volt-hours required to complete a run must be determined empirically. A more complex sample in terms of number of proteins or even a different sample buffer might require increased volt-hours. The time needed to achieve the programmed volt-hours depends on the pH range of the IPG strip used as well as sample and buffer characteristics. If different strips are run at the same time, the electrical conditions experienced by individual strips will be different, perhaps exposing some strips to more current than desired, since the total current limit is averaged over all

Second-dimension separation is by protein mass, or MW, using SDS-PAGE. The proteins resolved in IPG strips in the first dimension are applied to second-dimension gels and separated by MW perpendicularly to the first dimension. The pores of the second-dimension gel sieve proteins according to size because dodecyl sulfate coats all proteins essentially in proportion to their mass. The net effect is that proteins migrate as ellipsoids with a uniform negative chargeto- mass ratio, with mobility related logarithmically to mass (Garfin 1995).

Homogeneous (single-percentage acrylamide) gels generally give excellent resolution of sample proteins that fall within a narrow MW range. Gradient gels have two advantages: The percentage of acrylamide, often referred to as %T (total percentage of acrylamide plus crosslinker) determines the pore size of a gel. Most protein separations use 37.5 parts acrylamide to 1 part bis-acrylamide (bis). Some researchers substitute piperazine bisacrylamide (PDA), which can reduce silver staining background and give higher gel strength. If the total percentage of acrylamide plus crosslinker is higher, the smaller is the pore size. A suitable %T can be estimated from charts of mobility for proteins of different MW.
