Twodimensional Polyacrylamide Gel Electrophoresis

and the second gel dimension separated proteins according to their sizes. Thus, peptides are separated from one another according to two independent biochemical properties. 2D-PAGE was shown to be particularly valuable in the study, as well as in the identification of thousands of cellular or secreted proteins, including many of those present in human plasma/serum (Figure 1).

During the past few years, tremendous progress has been made in the field of 2D-PAGE. The 2D technique has been simplified, and, more importantly, made reproducible. Commercially manufactured im mobilized pH gradients (IPGs), with both acidic and basic high resolution power and precast sodium dodecyl sulfate (SDS) PAGE gels are now available. In addition, progress in protein solubilization and in the development of systems allowing high loading capacities has been made.

More than 20 years after its birth, 2D-PAGE is now a major technique in protein sciences. Over the past few years there has been an exponential increase in the creation and expansion of protein databases such as the swiss-2DPAGE, the heart-2dpage and the hsc-2dpage. Furthermore, tools have been developed to

Figure 1 The normal human plasma 2D map. Polypeptides (0.3 ^L of plasma) were separated by pH 3.5-10 carrier ampholyte gradient, followed by gradient 9-16% T polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The ammoniacal silver-stained gel was photographed with the higher molecular weight at the top and the acidic side on the left. 1, a2-macroglobulin; 2, ceruloplasmin; 3, glu-plasminogen; 4, lys-plasminogen; 5, complement factor B; 6, complement C1s; 7, protrans-ferrin; 8, prothrombin; 9, arB-glycoprotein; 10, transferrin; 11, hemopexin; 12, a2-antiplasmin; 13, a^antichymotrypsin; 14, fibrinogen a chain; 15, a2-HS-glycoprotein; 16, arantitrypsin; 17, antithrombin III; 18, fibrinogen p chain; 19, extended fibrinogen y chain; 20, Ge-globulin; 21, lysin-rich glycoprotein; 22, fibrinogen y chain; 23, apolipoprotein A-IV; 24, haptoglobin p chain; 25, Zn-a-glycoprotein; 26, apolipoprotein J; 27, cleaved haptoglobin p chain; 28, apolipoprotein E (phenotype E 3/4); 29, y chain of complement C4; 30, armicroglobulin; 31, apolipoprotein D; 32, apo A-I; 33, proapolipoprotein A-I; 34, retinol-binding protein; 35, haptoglobin a, chain; 36; transthyretin (prealbumin); 37, haptoglobin a2 chain; 38, haemoglobin p chain; 39, apolipoproteins C-II and C-III; a, albumin; pi, polyclonal heavy chains of IgM; a, polyclonal heavy chains of IgA; y, polyclonal heavy chains of IgG; k-X, polyclonal Ig light chains. Reproduced with permission from Tissot and Spertini (1995).

Figure 1 The normal human plasma 2D map. Polypeptides (0.3 ^L of plasma) were separated by pH 3.5-10 carrier ampholyte gradient, followed by gradient 9-16% T polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The ammoniacal silver-stained gel was photographed with the higher molecular weight at the top and the acidic side on the left. 1, a2-macroglobulin; 2, ceruloplasmin; 3, glu-plasminogen; 4, lys-plasminogen; 5, complement factor B; 6, complement C1s; 7, protrans-ferrin; 8, prothrombin; 9, arB-glycoprotein; 10, transferrin; 11, hemopexin; 12, a2-antiplasmin; 13, a^antichymotrypsin; 14, fibrinogen a chain; 15, a2-HS-glycoprotein; 16, arantitrypsin; 17, antithrombin III; 18, fibrinogen p chain; 19, extended fibrinogen y chain; 20, Ge-globulin; 21, lysin-rich glycoprotein; 22, fibrinogen y chain; 23, apolipoprotein A-IV; 24, haptoglobin p chain; 25, Zn-a-glycoprotein; 26, apolipoprotein J; 27, cleaved haptoglobin p chain; 28, apolipoprotein E (phenotype E 3/4); 29, y chain of complement C4; 30, armicroglobulin; 31, apolipoprotein D; 32, apo A-I; 33, proapolipoprotein A-I; 34, retinol-binding protein; 35, haptoglobin a, chain; 36; transthyretin (prealbumin); 37, haptoglobin a2 chain; 38, haemoglobin p chain; 39, apolipoproteins C-II and C-III; a, albumin; pi, polyclonal heavy chains of IgM; a, polyclonal heavy chains of IgA; y, polyclonal heavy chains of IgG; k-X, polyclonal Ig light chains. Reproduced with permission from Tissot and Spertini (1995).

compare 2D gels images across the Internet. Most importantly, methods for the analysis of 2D gels are continually improving. The high sensitivity and throughput of these techniques now enable the characterization of hundreds of proteins from a whole cell, tissue or body fluid. Proteins can be identified according to primary parameters such as their isoelec-tric points, apparent molecular mass, real mass and protein N- or C-terminal sequence tag, but also according to secondary attributes such as peptide mass fingerprint, peptide fragmentation data or amino acid composition. Interfacing and integrating databases from 2D gels such as swiss-2DPAGE, swiss-prot, GenBank, EMBL nucleotide sequence database, dbest, GSBD and the NLM's medline bibliographical reference database provide researchers with invaluable tools to study both genome and proteome. The continuing progress accomplished in both proteome research and bio-information will contribute to the implement of the Cyber-Encyclopedia of the Pro-teome, as suggested by R.D. Appel. This development increases the need for simple protocols to run reproducible 2D gels and is an important step for investigators involved in proteomics. several well-written protocols and reviews are accessible in the literature or through the World Wide Web (http://expasy.hcuge. ch/ch2d/technical-info.html; http://www.abdn.ac. uk/&mmb023/2dhome.htm). We will review here some of the important features that must be evaluated in order to implement a new 2D-gel laboratory.

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