Rapid high resolution two‐dimensional electrophoresis of human sperm proteins

Abstract

An improved protocol has been developed for two-dimensional gel analysis of human sperm proteins through the application of recent technical advances. Advantages of this protocol consist of increased resolving power, reduced processing time, enhanced reproducibility of staining patterns, and applicability to small quantities of protein. Technical improvements include determination of optimal protein loading (20 micrograms/gel), development of a reliable tube gel casting system, and application of minigel technology. In this study over 500 proteins were resolved with molecular weights ranging from 12,000 to 105,000 and isoelectric points from 5.0 to 8.5. This is a three-fold improvement in resolution over earlier results. A single operator was able to generate two-dimensional gels of multiple samples in less than one day. This rapid high resolution technique should facilitate further investigation of human sperm proteins under normal and pathologic conditions.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors

“…In 1990, Naaby-Hansen et al [2] acquired an electrophoretic map of acidic and neutral human spermatozoal proteins and obtained over 260 protein spots. In 1992, Kritsas et al [3] obtained over 500 spermatozoal protein spots with molecular mass ranging from 12 to 105 k and isoelectric points from 5.0 to 8.5. In 1994, Xu et al [4] acquired more than 600 spermatozoal protein spots with molecular mass ranging from 7.9 to 93.5 k and isoelectric points between 4.0 and 7.0.…”

Two-Dimensional Gel Electrophoretic Analysis of Vectorially Labeled Surface Proteins of Human Spermatozoa1

“…Specialized computer software has facilitated the quantitative analysis of 2D gels, including the ability to compare various computer images, which permits a detailed description of alterations in protein patterns resulting from experimental manipulations or changing environmental stimuli [13][14][15][16][17]. Although 2D electrophoresis has been employed in the study of human sperm proteins in the past [18][19][20][21][22][23][24][25][26], the present study was undertaken because reconciliation of the protein patterns reported in these previous reports was difficult because of the lack of standard procedures. Further studies were also needed to serve as the basis for a comprehensive cataloging of human sperm surface proteins because available data were limited in one or more of the following ways: 1) resolution, 2) reproducibility, 3) pH range, 4) specificity of surface labeling techniques, or 5) plasma membrane purification procedures.…”