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

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.

Towards complete analysis of the platelet proteome

“…Consequently, a range of 2-DE protein maps has been published using different protein detection methods and addressing different cellular compartments. As much insight into platelet biology has been gained by these studies the number of proteins identified which are expressed in platelets and annotated on 2-DE protein maps is still very small [9][10][11]. The most comprehensive maps are those from the interactive SWISS-PROT 2DPAGE, which gives information on less than 50 protein spots (http://www.expasy.ch/ch2d/), and from a 3-10 2-DE map recently published by Marcus et al with 186 identified protein features [12].…”

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

“…With the tube gels used for IPG, these problems are expected to be overcome. Although apparatus to prepare IPG tube gels had been developed previously [2,3], further devices were necessary to prepare gels in tubes efficiently.…”

“…Apparatus for preparing IPG tube gels have been developed as described [2,3]. Hochstrasser et al [2] used a special multichannel pump to transfer the gel solution into the capillary tubes.…”

“…There was no difference in resolution of proteins between 3 mm ID and 1.5 mm ID gels. Asakawa [3] established a technique to prepare IPG tube gels; he prepared the gels in the tubes held in a larger siliconized glass tube. However, since a large amount of useless gel solution was polymerized between the gel tubes and between the gel tube and the holder tube, one could lose a not-sonegligible amount of gel solution; also, it was often troublesome to remove the tubes stuck to the holder with polyacrylamide gels after polymerization.…”