Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes.
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.
Asakawa
J
J
Department of Genetics, Radiation Effects Research Foundation, Hiroshima, Japan.
D001792:Blood Platelets / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008970:Molecular Weight; D010455:Peptides / Q000097:blood* / Q000302:isolation & purification
D007202:Indicators and Reagents; D010455:Peptides