**2.1 Experimental design**

#### **2.1.1 Sample preparation**

Efficient and reproducible sample preparation methods are a key to successful 2-DE (Rabilloud 1999, Macri *et al.* 2000, Molloy 2000). Sample preparation methods range from extraction with simple solubilization solutions to complex mixtures of chaotropic agents, detergents, and reducing agents. Sample preparation can include enrichment strategies for separating protein mixtures into reproducible fractions.

An effective sample preparation procedure will:


Two Dimensional Gel Electrophoresis in Cancer Proteomics 363

to maintain the solubility of some proteins. Salt should be present in a sample only if it is an absolute requirement, and then only at a total concentration less than 40 mM. This is problematic since any salt included will be removed during the initial high-current stage of focusing. Salt limits the voltage that can be achieved without producing high current, increasing the time required for focusing. Proteins that require salt for solubility are subject to precipitation once the salt is removed. Carrier ampholytes sometimes help to counteract insufficient salt in a sample. They are usually included at a concentration of ≤0.2% (w/v) in sample solutions for IPG strips. High concentrations of carrier ampholytes will slow down IEF until they are focused at their pI, since they carry current and hence limit voltage. Some researchers have increased resolution by varying the ampholyte

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

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,

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

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

solved this problem by supplying it in a form safe for shipping and lab use.

Nilsson *et al.* 2000) have been used to reduce the complexity of samples.

composition.

**2.1.6 Reducing agents** 

**2.1.7 Prefractionation** 

**2.1.8 Removal of albumin and IgG** 

plasma samples prior to 2-DE.

**2.1.9 Sequential extraction** 


Most protein mixtures will require some experimentation to determine optimum conditions for 2-D PAGE. Variations in the concentrations of chaotropic agents, detergents, ampholytes, and reducing agents can dramatically affect the 2-D pattern.

#### **2.1.2 Solubilization**

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, Rabilloud 2000).

#### **2.1.3 Chaotropic agents**

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 chaotropic conditions are required (Rabilloud 1998).

#### **2.1.4 Detergents**

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

#### **2.1.5 Carrier ampholytes**

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 to maintain the solubility of some proteins. Salt should be present in a sample only if it is an absolute requirement, and then only at a total concentration less than 40 mM. This is problematic since any salt included will be removed during the initial high-current stage of focusing. Salt limits the voltage that can be achieved without producing high current, increasing the time required for focusing. Proteins that require salt for solubility are subject to precipitation once the salt is removed. Carrier ampholytes sometimes help to counteract insufficient salt in a sample. They are usually included at a concentration of ≤0.2% (w/v) in sample solutions for IPG strips. High concentrations of carrier ampholytes will slow down IEF until they are focused at their pI, since they carry current and hence limit voltage. Some researchers have increased resolution by varying the ampholyte composition.
