**2.1.6 Reducing agents**

362 Gel Electrophoresis – Advanced Techniques

5. Yield proteins of interest at detectable levels, which may require the removal of

Most protein mixtures will require some experimentation to determine optimum conditions for 2-D PAGE. Variations in the concentrations of chaotropic agents, detergents,

Solubilization of proteins is achieved by the use of chaotropic agents, detergents, reducing agents, buffers, and ampholytes. These are chosen from a small list of compounds that meet the requirements, both chemically and electrically, for compatibility with the technique of IEF in IPG strips. The compounds chosen must not increase the ionic strength of the solution, to allow high voltages to be applied during focusing without producing high currents. Thorough discussion of solubilization methods, including new variations, can be found in several books (Pennington and Dunn 2001,

Urea is the most commonly used chaotropic agent in sample preparation for 2-D PAGE. Thiourea can be used to help solubilize many otherwise intractable proteins. Urea and thiourea disrupt hydrogen bonds and are used when hydrogen bonding causes unwanted aggregation or formation of secondary structures that affect protein mobility. Urea is typically used at 8 M. Thiourea is weakly soluble in water, but is more soluble in high concentrations of urea, so a mixture of 2 M thiourea and 5–8 M urea is used when strongly

Detergents are added to disrupt hydrophobic interactions and increase solubility of proteins at their pI. Detergents must be nonionic or zwitterionic to allow proteins to migrate according to their own charges. Some proteins, especially membrane proteins, require detergents for solubilization during isolation and to maintain solubility during focusing. Ionic detergents such as SDS are not compatible with IEF, but can be used with concentrated samples in situations where the SDS can be unbound from the proteins by IEF-compatible detergents that compete for binding sites. Nonionic detergents such as octylglucoside, and zwitterionic detergents such as CHAPS and its hydroxyl analog, CHAPSO, can be used. CHAPS, CHAPSO, or octylglucoside concentrations of 1–2% are recommended (Rabilloud 1999). New detergents are emerging that have great potential in proteomics, including SB 3- 10 and ASB-14 (Chevallet *et al.* 1998). Some proteins may require detergent concentrations as

A fundamental challenge with IEF is that some proteins tend to precipitate at their pI. Even in the presence of detergents, certain samples may have stringent salt requirements

4. Remove or thoroughly digest nucleic acids and other interfering molecules

interfering abundant proteins or nonrelevant classes of proteins

ampholytes, and reducing agents can dramatically affect the 2-D pattern.

**2.1.2 Solubilization** 

Rabilloud 2000).

**2.1.4 Detergents** 

**2.1.3 Chaotropic agents** 

chaotropic conditions are required (Rabilloud 1998).

high as 4% for solubility (Hermann *et al.* 2000).

**2.1.5 Carrier ampholytes** 

Reducing agents such as dithiothreitol (DTT) or tributylphosphine (TBP) are used to disrupt disulfide bonds. Bond disruption is important for analyzing proteins as single subunits. DTT is a thiol reducing agent added in excess to force equilibrium toward reduced cysteines. At 50 mM it is effective in reducing most cystines, but some proteins are not completely reduced by this treatment. If the concentration of DTT is too high it can affect the pH gradient since its pKa is around 8. TBP is a much more effective reducing agent than DTT. It reacts to reduce cystines stoichiometrically at low millimolar concentrations (Herbert *et al.* 1998). It is chemically more difficult to handle than DTT, but Bio-Rad has solved this problem by supplying it in a form safe for shipping and lab use.

#### **2.1.7 Prefractionation**

Reducing the complexity of the sample loaded on a 2-D gel can increase the visibility of minor proteins. Techniques such as differential extraction (Molloy *et al.* 1998), subcellular fractionation (Taylor *et al.* 2000, Morel *et al.* 2000), chromatography (Fountoulakis *et al.* 1999), or prefocusing in a preparative IEF device such as the Rotofor® system (Masuoka *et al.* 1998, Nilsson *et al.* 2000) have been used to reduce the complexity of samples.

#### **2.1.8 Removal of albumin and IgG**

The isolation of lower-abundance proteins from serum or plasma is often complicated by the presence of albumin and immunoglobulin G (IgG). Albumin is the most abundant protein (~60–70%) in serum and IgG is the second most abundant protein (10–20%). These two proteins effectively act as major contaminants, masking the presence of many co-migrating proteins, as well as limiting the amount of total serum protein that can be resolved on a 2-D gel. In the past, removal of albumin and IgG usually required separate chromatography methodologies for each of the two species. Now, Bio-Rad's Aurum™ serum protein kit allows selective binding and simultaneous removal of both albumin and IgG from serum or plasma samples prior to 2-DE.

#### **2.1.9 Sequential extraction**

One method for reducing sample complexity is the basis of the ReadyPrep™ sequential extraction kit. This protocol takes advantage of solubility as a third independent means

Two Dimensional Gel Electrophoresis in Cancer Proteomics 365

types of charged groups in a protein. Proteins are amphoteric molecules. As such, they can carry positive, negative, or zero net charge depending on the pH of their local environment. For every protein there is a specific pH at which its net charge is zero; this is its pI. Proteins show considerable variation in pI, although pI values usually fall in the range of pH 3–12, with the majority falling between pH 4 and pH 7. A protein is positively charged in solution

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

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

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

at pH values below its pI and negatively charged at pH values above its pI.

state positions from anywhere in the system.

**2.2.2 IEF** 

**2.2.3 IPG strips** 

(Righetti 1990).

**2.2.4 Choice of pH gradient ranges** 

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 *et al.* 1998).
