Immunoblotting, also dubbed Western blotting, exploits antibody specificity to identify target proteins among a number of unrelated protein species (1,2). Traditionally used for mapping known proteins on electrophoretically resolved mixtures, in the proteome era immunoblotting has been combined with two-dimensional (2D) gel electrophoresis for the rapid visualization and identification of target proteins (3), protein adducts with drugs (4), and antigenic proteins associated to pathogens, allergens as well as tumors, with patient serum used as primary antibody (5-9).

In the postgenomic era, gene expression is becoming a focus of attention and it is widely accepted that one gene does not necessarily encode a single protein product because molecular mechanisms generating different isoforms from the same gene have been described in many different organisms. Although the relation between number of genes and number of potentially encoded proteins has yet to be clarified, it is usually recognized that the number of polypeptides expressed by a genome is greater than expected on the basis of calculated gene numbers. In fact, it has been estimated that the average number of alternates spliced from the transcript of a single mammalian gene may be two to three or more (10) and that many proteins may be subject to co- and posttranslational modifications and proteolytic processing. For example, up to 20% of protein is acetylated in yeast (11). Several preliminary proteome projects have also shown that prokaryotic organisms can express more than one protein isoform from a single gene and posttranslationally modified gene products have been reported (12). Moreover, compared with completely sequenced genomes of other organisms, the unexpected low number of potential protein-coding genes in the human genome makes molecular mechanisms generating different isoforms from the same gene particularly interesting. As shown in Scheler et al. (13) and Janke et al. (14), simultaneous 2D gel analysis of cross-reactive protein isoforms derived from a single gene may produce very complex isoforms patterns. More than 50 and 80 2D electro-phoretic protein spots where observed using specific antibodies for

HSP27 and Tau protein, respectively. 2D immunoblotting combined with mass spectrometry-based identification methods has been widely applied to the characterization of 2D electrophoretic cross-reactive isoforms of the same protein, e.g., resulting from alternative splicing, co- and/or posttranslational modifications and proteolytic cleavages (15-19).

Western blotting analysis is also the method of choice for simultaneous visualization of 1D- and 2D-separated proteins sharing common epitopes related to specific posttranslationally modified amino acids or to specific functional/structural domains, for example, antiphosphoresidues and anti-O-linked V-acetylgluco-samine antibodies (20-22). This approach has been widely used to differentially visualize tyrosine-phosphorylation profiles in cells and tissues under different conditions (17,20), as well as for studying signal transduction pathways following stimulation (23). The dynamic and reversible nature of several known posttranslational modifications makes their characterization possible only at protein level, as these features characterize mature gene products and cannot be inferred from crude genome-derived amino acid sequences stored in sequence databases. Two-dimensional immunoblotting is therefore a powerful method for rapid visualization of target proteins, sharing a common feature, to be identified and characterized by microchemical analysis, for example, by mass spectrometry.

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ("blotted") onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds.

A simplified procedure in which protein samples are not separated electrophoretically but are spotted directly onto the membrane is called "dot blot" and is a good preliminary technique for the detection of an antigen in a sample.

A number of related techniques for probing membranes containing transferred protein with specific ligands has been described:

• In "Far-Western blotting" the membrane is probed with another protein to detect specific protein-protein interactions (24). The reaction can be revealed using biotinylated or GST-tagged bait or "probe" protein followed by a streptavidin-HRP or an anti-GST-HRP chemi-luminescent detection system, respectively.

• Blot overlays include the probing of membrane with various molecules to detect the presence of specific binding domains, for example, with guanine triphosphate (25,26) or proteoglycans (27). In the Southern or North Western blotting, the membrane is probed with deoxyribonucleic acid or ribonucleic acid molecules to detect nucleic-acid binding proteins (28).

• In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden).

2. Materials

2.1. Equipment

1. Blotting apparatus: transfer cell, gel holder, magnetic stirrer, refrigerated thermostatic circulator unit.

2. Power supply.

3. Rocking agitator.

4. Computing Densitometer and/or gel and blot image acquisition system.

5. PC with a computer program for 2D gel analysis.

2.2. Reagents

1. Distilled water.

2. Nitrocellulose membrane.

3. Transfer buffer: 25 ml Tris-HCl, 192 mM glycine, 20% (v/v) methanol. Do not adjust pH; it is approx 8.3.

4. Filter paper for blotting (Whatman 17 Chr).

5. Ponceau S solution: 0.2% (w/v) Ponceau S in 3% (w/v) trichloroace-tic acid.

6. Coomassie blue solution: Coomassie blue R-250 0.1% in 40% methanol, 1% acetic acid.

7. Destaining solution: 50% methanol.

8. Phosphate-buffered saline (PBS): 0.15 M NaCl, 10 mM NaH2PO4; bring to pH 7.4 with NaOH.

9. Blocking solution: 3% (w/v) non-fat dry milk in PBS, Triton X-100 0.1% (w/v).

10. Primary antibody solution: primary antibody, appropriately diluted in blocking solution.

11. Secondary antibody solution: secondary antibody, appropriately diluted in blocking solution.

12. Washing solution: Triton X-100 0.5% (w/v) in PBS.

14. Amersham Biosciences ECL (enhanced chemiluminescence) kit, cat. no. RPN 2106.

15. Saran Wrap® or other cling-films.

16. X-ray films, 18 x 24 cm (Amersham Hyper film ECL; cat. no. RPN 3103).

17. Developer and fixer for X-ray film (Developer replenisher; fixer and replenisher, 3M, cat. nos. XAF 3 and XAD 3; 3M Italia S.p.A., Segrate, Italy).

18. Stripping buffer: 100 mM 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate (SDS), 62.5 mM Tris-HCl, pH 6.8.

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