Horizontal Two-dimensional Electrophoresis Research Articles

Glycolytic enzyme expression in human granulosa cells

To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. Laboratory study. University Hospital. Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.

Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems

Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems

Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems.

We have developed a simplified procedure for the separation of enzyme-digested genomic DNA, or of complex mixtures of cloned DNA, into two dimensions. The procedure relies on the use of precast gels for horizontal electrophoretic separations. Precast agarose-type gels are used for the first-dimensional separation of fragments based on size. Precast polyacrylamide gels are used for the second-dimensional separation of fragments. The separated fragments are subjected to enzymatic digestion in situ prior to their transfer to the second-dimensional gel. Applications of this procedure include the analysis of DNA libraries, analysis of yeast artificial chromosomes (YACs) as well as other similar preparations, and the screening of genomic DNA for the occurrence of multi-copy DNA fragments as in the case of genomic amplifications in cancer.

Horizontal two‐dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension

A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of CSF samples. This high-resolution method is a tool worthy of consideration for the research of CSF proteins and disease-specific changes in different neurological disorders.

Analysis of porcine erythrocyte lysate proteins by horizontal two-dimensional electrophoresis with immobilized pH gradient in the first dimension.

Horizontal two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension resulted in excellent patterns of porcine erythrocyte lysate proteins. The overwhelming excess of hemoglobin was removed by chloroform-ethanol extraction and the number of spots further increased by acetone precipitation, revealing considerably more protein spots than detected by analyzing the untreated sample. A total of 430 protein spots have been reproducibly obtained for further analysis of genetic variations. A number of enzyme systems have been identified by visual inspection and comparison with previous results. The existence of further multiple forms for other enzymes is also apparent.

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension.

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.

Horizontal two-dimensional electrophoresis with immobilized pH gradients using PhastSystem.

Protocols for horizontal two-dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two-dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first-dimensional immobilized pH gradient gel strip prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evaluated. Silver stained two-dimensional patterns were obtained within 3.5 h.