Odile Carrette

State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. Here we provide comprehensive step-by-step instructions for the application of a standardized 2D-PAGE protocol to a sample of human plasma or cerebrospinal fluid (CSF). The method can be easily adapted to any type of sample. This four-day protocol provides detailed information on how to apply complex biological fluids to an immobilized dry strip gel, cast home-made gradient acrylamide gels, run the gels, and perform standard staining methods. A troubleshooting guide is also included.NOTE:The version of this article initially published online contained the following errors in the REAGENT SETUP section on p. 814: Under “Strip reswelling buffer”, “0.0375 g of DTE” should be “0.0385 g of DTE”. Under “Iso Buffer”, “5.4 g of urea” should be “4.8 g of urea”. Under “Electrophoresis buffer, upper tank”, “18 g of Tris” should be “9.09 g of Tris” and “86.4 g of glycine” should be “44.6 g of glycine”. Under “Electrophoresis buffer, lower tank”, “90 g of Tris” should be “45.45 g of Tris” and “432 g of glycine” should be “222.9 g of glycine”. Under “Agarose sealing solution”, “5%” should be “0.5%”. Under “Sensitizer solution”, “68.4 g of sodium acetate” should be “68.04 g of sodium acetate trihydrate”. Under “Ammoniacal silver nitrate solution”, “Silver nitrate” should be “47 mM silver nitrate”; “10 N sodium hydroxide” should be “0.02 N sodium hydroxide”; and “25% ammonium hydroxide (27% pure, Merck)” should be 0.33% ammonium hydroxide (25% pure, Merck)”. The sentence “Add 6 g of silver nitrate dissolved in dH2O to 30ml slowly into a solution containing 160 ml of dH2O, 10 ml of concentrated ammonia (25%) and 1.5 ml of 10 N sodium hydroxide” should read “Dissolve 6 g of silver nitrate in dH2O to 30 ml, then add slowly into a solution containing 160 ml of dH2O, 10 ml of 25% ammonium hydroxide and 1.5 ml of 10 N sodium hydroxide.” In Table 1 (p. 816), “Up to 100,000 kVh” should be “100kVh”. These errors have been corrected in all versions of the article.