**2.2.2 IEF**

364 Gel Electrophoresis – Advanced Techniques

of protein separation. Proteins are sequentially extracted in increasingly powerful solubilizing solutions. More protein spots are resolved by applying each solubility class to a separate gel, thereby enriching for particular proteins while simplifying the 2-D patterns in each gel. An increase in the total number of proteins is detected using this approach (Molloy

The presence of nucleic acids, especially DNA, interferes with separation of proteins by IEF. Under denaturing conditions, DNA complexes are dissociated and markedly increase the viscosity of the solution, which inhibits protein entry and slows migration in the IPG. In addition, DNA binds to proteins in the sample and causes artifactual migration and streaking. The simplest method for removal of DNA is enzymatic digestion. Adding endonuclease to the sample after solubilization at high pH (40 mM Tris) allows efficient digestion of nucleic acids while minimizing the action of contaminating proteases. The advantage of the endonuclease method is that sample preparation can be achieved in a

The amount of protein applied to an IPG strip can range from several micrograms to 1 mg or more (Bjellqvist *et al.* 1993a). Some of the factors affecting the decision of how much protein

a. Subsequent analysis. Enough of the protein of interest must be loaded for it to be analyzed. With the Ready Gel® mini system (7 cm IPG), detection of moderately abundant proteins in complex mixtures with Coomassie Brilliant Blue R-250 dye requires on the order of 100 μg total protein. With the same load, many low-abundance proteins can be detected with more sensitive stains such as silver or SYPRO Ruby protein gel stain. b. The purpose of the gel. If the gel is being run solely for the sake of getting a good image of well-resolved proteins for comparative studies or for publication, the protein load

c. The abundance of the proteins of interest. If the purpose is to study low-copy-number proteins, a large mass of a protein mixture might be loaded (Wilkins *et al.* 1998). d. The complexity of the sample. A highly complex sample containing many proteins of widely varying concentrations might require a compromise load so that highabundance proteins don't obscure low-abundance proteins. By enriching a sample for specific types of proteins using prefractionation techniques, each individual protein will be at a higher relative concentration, which means that enough material can be loaded

e. pH range of IPG strip. In general, larger amounts of total protein can be loaded on a narrow-range IPG strip. Only the proteins with a pI within the strip pH range will be

Differences in proteins' pI are the basis of separation by IEF. The pI is defined as the pH at which a protein will not migrate in an electric field and is determined by the number and

single step, by the addition of the enzyme prior to loading the first-dimension IPG.

would be the minimum amount that is stainable.

for detection of low-abundance constitutents.

represented within the second-dimension gel.

**2.2 The first dimension: Isoelectric Focusing (IEF)** 

**2.2.1 Isoelectric point (pI)** 

*et al.* 1998).

**2.1.10 Removal of DNA** 

**2.1.11 Protein load** 

to load are:

When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose protons. As it migrates, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equal to its pI. There, being uncharged, it will stop migrating. If this protein should happen to diffuse to a region of lower pH, it will become protonated and be forced back toward the cathode by the electric field. If, on the other hand, it diffuses into a region of pH greater than its pI, the protein will become negatively charged and will be driven toward the anode. In this way, proteins condense, or are focused, into sharp bands in the pH gradient at their individual characteristic pI values. Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pI values at differing rates but remain relatively fixed at those pH values for extended periods. By contrast, proteins in conventional electrophoresis continue to move through the medium until the electric field is removed. Moreover, in IEF, proteins migrate to their steady state positions from anywhere in the system.

#### **2.2.3 IPG strips**

A stable, linear, and reproducible pH gradient is crucial to successful IEF. IPG strips offer the advantage of gradient stability over extended focusing runs (Bjellqvist *et al.* 1982). IPG strips are much more difficult to cast than carrier ampholyte gels (Righetti 1983); however, IPG strips are commercially available, for example as ReadyStrip™ IPG strips. pH gradients for IPG strips are created with sets of acrylamido buffers, which are derivatives of acrylamide containing both reactive double bonds and buffering groups. The general structure is CH2=CH–CO–NH–R, where R contains either a carboxyl [–COOH] or a tertiary amino group (e.g., N (CH3)2). These acrylamide derivatives are covalently incorporated into polyacrylamide gels at the time of casting and can form almost any conceivable pH gradient (Righetti 1990).

#### **2.2.4 Choice of pH gradient ranges**

Use of broad-range strips (pH 3–10) allows the display of most proteins in a single gel. With narrow-range and micro-range overlapping gradient strips, resolution is increased by expanding a small pH range across the entire width of a gel. Since many proteins are focused in the middle of the pH range 3–10, some researchers use nonlinear (NL) gradients to better resolve proteins in the middle of the pH range and to compress the width of the extreme pH ranges at the ends of the gradients. However, overlapping narrow-range and micro-range linear IPG strips can outperform a nonlinear gradient and display more spots

Two Dimensional Gel Electrophoresis in Cancer Proteomics 367

the gel matrix under current as well as by absorption. The PROTEAN IEF cell has preprogrammed methods designed to accommodate active rehydration. Active rehydration is thought to help large proteins enter the strip by applying electrical "pull". Because the voltage is applied before all the solution and proteins are absorbed into the gel, the pH of a protein's environment will be the pH of the rehydration buffer, and the protein will move according to its mass-to charge ratio in that environment. Thus, small proteins with a higher mobility have a higher risk of being lost from the strip. With passive rehydration, proteins enter the gel by absorption only. This method allows efficient use of equipment since strips can be rehydrated in sample rehydration trays while other samples are being focused in the

Whether the strips are hydrated actively or passively, it is very important that they be incubated with sample for at least 11 hr prior to focusing. This allows the high molecular weight proteins time to enter the gel after the gel has become fully hydrated and the pores have attained full size. These sample application methods work because IEF is a steady-state

b. Sample application during rehydration avoids the problem of sample precipitation,

c. Shorter focusing times can be used because the sample proteins are in the IPG strip

Cup loading can be beneficial in the following cases (Cordwell *et al.* 1997, Görg *et al.* 2000): When samples contain high levels of DNA, RNA, or other large molecules, such as

Because of its relative difficulty and tendency toward artifacts, cup loading should be avoided if possible. When loading the protein sample from a cup, the IPG strips must be rehydrated

The rehydration tray is recommended although IPG strips are often rehydrated in 1 or 2 ml pipettes that have been sealed at both ends with Para film. Sample volumes of up to 100 μl

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.

For analytical serum samples that have not been treated to remove albumin

prior to sample application. The IPG strips can be rehydrated in a variety of ways.

technique, so proteins migrate to their pI independent of their initial positions.

IEF cell.

The advantages of this approach are:

prior to IEF

cellulose

a. Sample application is simple (Görg *et al.* 1999)

**2.2.9 Sample application by cup loading** 

When running basic IPG strips; e.g., pH 7–10

which often occurs with cup loading (Rabilloud 1999)

d. Very large amounts of protein can be loaded using this method

For samples that contain high concentrations of glycoproteins

can be loaded later onto each gel strip using a sample cup.

**2.2.10 Power conditions and resolution in IEF** 

per sample. This result is due to the extra resolving power from use of a narrower pI range per gel. Use of overlapping gradients also allows the ability to create "cyber" or composite gels by matching spots from the overlapping regions using imaging software.
