**2.4.1 Guidelines for detection of proteins in gels**

Gels are run for either analytical or preparative purposes. The intended use of the gel determines the amount of protein to load and the means of detection. It is most common to make proteins in gels visible by staining them with dyes or metals. Each type of protein stain has its own characteristics and limitations with regard to the sensitivity of detection. Sometimes proteins are transferred to membranes by western blotting to be detected by immunoblotting, glycoprotein analysis, or total protein stain. If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract, or contaminants in a purification scheme), then a high protein load (0.1–1 mg/ml) and a high-sensitivity stain, such as silver or a fluorescent stain, should be used (Corthals *et al.* 2000). When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix proteins in the gel. Quantitative comparisons require the use of stains with broad linear ranges of detection. The sensitivity that is achievable in staining is determined by: 1) the amount of stain that binds to the proteins; 2) the intensity of the coloration; 3) the difference in coloration between stained 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 with high-purity water between stains.

### **2.4.2 Coomassie blue staining**

370 Gel Electrophoresis – Advanced Techniques

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

The migration rate of a polypeptide in SDS-PAGE is inversely proportional to the logarithm of its MW. The larger the polypeptide, the more slowly it migrates in a gel. MW is determined in SDS-PAGE by comparing the migration of protein spots to the migration of standards. Plots of log MW versus the migration distance are reasonably linear. Gradient SDS-PAGE gels can also be used to estimate MW. In this case, log MW is proportional to log (%T). With linear gradients, %T is proportional to distance migrated, so the data can be plotted as log MW vs. log (migration distance). Standard curves are actually sigmoid. The apparent linearit of a standard curve may not cover the full MW range for a given protein mixture in a particular gel. However, log MW varies sufficiently slowly to allow accurate MW estimates to be made by interpolation, and even extrapolation, over relatively wide ranges (Garfin 1995). Mixtures of standard proteins with known MW are available from Bio-Rad in several formats for calibrating the migration of proteins in electrophoretic gels. Standards are available unstained, prestained, or with tags for development with various secondary reagents (useful when blotting). Standards can be run in a reference well, attached to the end of a focused IPG strip by filter paper, or directly embedded in agarose

Gels are run for either analytical or preparative purposes. The intended use of the gel determines the amount of protein to load and the means of detection. It is most common to make proteins in gels visible by staining them with dyes or metals. Each type of protein stain has its own characteristics and limitations with regard to the sensitivity of detection. Sometimes proteins are transferred to membranes by western blotting to be detected by immunoblotting, glycoprotein analysis, or total protein stain. If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract, or contaminants in a purification scheme), then a high protein load (0.1–1 mg/ml) and a high-sensitivity stain, such as silver or a fluorescent stain, should be used (Corthals *et al.* 2000). When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix proteins in the gel. Quantitative comparisons require the use of stains with broad linear ranges of detection. The sensitivity that is achievable in staining is determined by: 1) the amount of stain that binds to the proteins; 2) the intensity of the coloration; 3) the difference in coloration between stained

**2.3.7 Second dimension and high throughput** 

and reproducibility.

**2.3.8 MW estimation** 

onto the second-dimension gel

**2.4 Detection of proteins in gels** 

**2.4.1 Guidelines for detection of proteins in gels** 

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

#### **2.4.3 SYPRO ruby fluorescent staining**

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 multiimager and VersaDoc™ imaging systems.

#### **2.4.4 Silver staining**

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

Two Dimensional Gel Electrophoresis in Cancer Proteomics 373

stain can be imaged with single-color and multicolor fluorescence via direct laser excitation. This system permits detection of almost any fluorophore that is excited in the visible spectrum. The internal laser and external laser options allow optimal excitation of singlecolor or multicolor fluorescent samples. Computer-controlled, user-accessible filter wheels have eight filter slots, allowing detection of many multicolor combinations of dyes (Gingrich

Computer-assisted image analysis software is an indispensable tool for the evaluation of

Image analysis systems deliver error-fee comprehensive qualitative and quantitative data from a large number of 2-D gels (Miller 1989). PDQuest software from Bio-Rad is a popular analysis tool. Gel analysis of digitized gel images includes spot detection, spot quantitation, gel comparison, and statistical analysis. PDQuest software has the further advantage of seamless integration with any of Bio-Rad's image acquisition instruments, as well as the ability to control the ProteomeWorks™ spot cutter. The advanced annotation feature can be

Before the software automatically detects the protein spots of a 2-D gel, the raw image data are corrected and the gel background is subtracted. The process is executed with simple menus and "wizards." PDQuest software models protein spots mathematically as 3-D Gaussian distributions and uses the models to determine absorption maxima. This enables automatic detection a resolution of merged spots. Following this procedure, spot intensities are obtained by integration of the Gaussian function. The mathematical description of the spots is used both for data reduction and for increasing evaluation speed, since reevaluation of data after an image change takes only fractions of a second. The hit rate of automatic spot detection is highly dependent on the quality of the 2-D gels. Correction capabilities of PDQuest software can be used to add undetected spots to the list of spots or to delete spots

The next step in 2-D gel evaluation is the identification of proteins that are present in all gels of a series. This task is made difficult primarily because of inherent irreproducibility in gels, which affects the positions of spots within a gel series. Gel analysis software must detect minor shifts in individual spot position within the gel series. Many software packages for automatic gel comparison are created with the assumption that the relative positions of spots are altered only slightly relative to each other, and allocate the spots on this basis. Prior to automatic gel comparison, PDQuest software selects the best 2-D gel of a gel series as a reference or standard gel and compares all other 2-D gels to this gel. Proteins in a gel

a. Storage and structuring of large amounts of collected experimental image data

used to label spots with text, URL links, document links, or mass spectrometry data.

**2.5.4 Computer-assisted image analysis of 2-D electrophoretic gels** 

b. Rapid and sophisticated analysis of experimental information

c. Supplementation and distribution of data among labs

d. Establishment of 2-D-protein data banks

**2.5.5 Spot detection and spot quantitation** 

*et al.* 2000).

complex 2-D gels. It allows:

that arise from gel artifacts.

**2.5.6 Gel comparison** 

heavily glycosylated proteins in gels. Protein spots containing 10–100 ng of protein can be easily seen. Proteins in gels are fixed with alcohol and acetic acid, then oxidized in a solution of potassium dichromate in dilute nitric acid, washed with water, and treated with silver nitrate solution. Silver ions bind to the oxidized proteins and are subsequently reduced to metallic silver by treatment with alkaline formaldehyde. Color development is stopped with acetic acid when the desired staining intensity has been achieved. This method is not compatible with mass spectroscopic analysis since the oxidative step changes protein mass. The Silver Stain Plus stain from Bio-Rad requires only one simultaneous staining and development step and is based on the method developed by Gottlieb and Chavko (1987). Proteins are fixed with a solution containing methanol, acetic acid, and glycerol, and washed extensively with water. The gels are then soaked in a solution containing a silverammine complex bound to colloidal tungstosilicic acid. Silver ions transfer from the tungstosilicic acid to the proteins in the gel by means of an ion exchange or electrophilic process. Formaldehyde in the alkaline solution reduces the silver ions to metallic silver to produce the images of protein spots. The reaction is stopped with acetic acid when the desired intensity has been achieved. Because silver ions do not accumulate in the bodies of gels, background staining is light. Since this method lacks an oxidizing step, visualization of heavily glycosylated proteins and lipoproteins can be less sensitive than with the Merril stain. This method is better for use in proteomics when the end goal is identification by mass spectrometric analysis.

#### **2.5 Image acquisition and analysis**

### **2.5.1 Image acquisition instruments**

Before 2-D gels can be analyzed with an image evaluation system, they must be digitized. The most commonly used devices are camera systems, densitometers, phosphor imagers, and fluorescence scanners. All of Bio-Rad's imaging systems are seamlessly integrated with PDQuest™ software, and they can export and import images to and from other software via TIFF files.

#### **2.5.2 Densitometry**

Densitometers compare the intensity of a light beam before and after attenuation by a sample. The GS-800™ calibrated imaging densitometer has been customized for analysis of gels, autoradiograms, and blots. The transmittance and true reflectance capabilities allow accurate scans of samples that are either transparent (gels and film) or opaque (blots). It provides high-quality imaging to resolve close spots and a variable resolution feature to preview and crop images. Wet 2-D gels may be scanned with red, green, and blue color CCD technology on the watertight platen.

#### **2.5.3 Storage phosphor and fluorescence scanners**

Digitization of 2-D gels stained with fluorescent dyes or radioactive compounds requires specific imaging systems (Patton 2000). The Molecular Imager FX™ Pro Plus system is flexible and expandable. 2-D gels of radiolabled proteins can be imaged using a Kodak phosphor screen more rapidly and accurately than with film. Popular proteomic fluorescent stains, including SYPRO Ruby protein gel and blot stains and SYPRO Orange protein gel

heavily glycosylated proteins in gels. Protein spots containing 10–100 ng of protein can be easily seen. Proteins in gels are fixed with alcohol and acetic acid, then oxidized in a solution of potassium dichromate in dilute nitric acid, washed with water, and treated with silver nitrate solution. Silver ions bind to the oxidized proteins and are subsequently reduced to metallic silver by treatment with alkaline formaldehyde. Color development is stopped with acetic acid when the desired staining intensity has been achieved. This method is not compatible with mass spectroscopic analysis since the oxidative step changes protein mass. The Silver Stain Plus stain from Bio-Rad requires only one simultaneous staining and development step and is based on the method developed by Gottlieb and Chavko (1987). Proteins are fixed with a solution containing methanol, acetic acid, and glycerol, and washed extensively with water. The gels are then soaked in a solution containing a silverammine complex bound to colloidal tungstosilicic acid. Silver ions transfer from the tungstosilicic acid to the proteins in the gel by means of an ion exchange or electrophilic process. Formaldehyde in the alkaline solution reduces the silver ions to metallic silver to produce the images of protein spots. The reaction is stopped with acetic acid when the desired intensity has been achieved. Because silver ions do not accumulate in the bodies of gels, background staining is light. Since this method lacks an oxidizing step, visualization of heavily glycosylated proteins and lipoproteins can be less sensitive than with the Merril stain. This method is better for use in proteomics

Before 2-D gels can be analyzed with an image evaluation system, they must be digitized. The most commonly used devices are camera systems, densitometers, phosphor imagers, and fluorescence scanners. All of Bio-Rad's imaging systems are seamlessly integrated with PDQuest™ software, and they can export and import images to and from other software via

Densitometers compare the intensity of a light beam before and after attenuation by a sample. The GS-800™ calibrated imaging densitometer has been customized for analysis of gels, autoradiograms, and blots. The transmittance and true reflectance capabilities allow accurate scans of samples that are either transparent (gels and film) or opaque (blots). It provides high-quality imaging to resolve close spots and a variable resolution feature to preview and crop images. Wet 2-D gels may be scanned with red, green, and blue color

Digitization of 2-D gels stained with fluorescent dyes or radioactive compounds requires specific imaging systems (Patton 2000). The Molecular Imager FX™ Pro Plus system is flexible and expandable. 2-D gels of radiolabled proteins can be imaged using a Kodak phosphor screen more rapidly and accurately than with film. Popular proteomic fluorescent stains, including SYPRO Ruby protein gel and blot stains and SYPRO Orange protein gel

when the end goal is identification by mass spectrometric analysis.

**2.5 Image acquisition and analysis 2.5.1 Image acquisition instruments** 

CCD technology on the watertight platen.

**2.5.3 Storage phosphor and fluorescence scanners** 

TIFF files.

**2.5.2 Densitometry** 

stain can be imaged with single-color and multicolor fluorescence via direct laser excitation. This system permits detection of almost any fluorophore that is excited in the visible spectrum. The internal laser and external laser options allow optimal excitation of singlecolor or multicolor fluorescent samples. Computer-controlled, user-accessible filter wheels have eight filter slots, allowing detection of many multicolor combinations of dyes (Gingrich *et al.* 2000).
