Two‐dimensional electrophoresis. Two‐dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

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Abstract
Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4–8 and 7–10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5–10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4–8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7–10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4–8 or 7–10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.