Search

Although two-dimensional electrophoresis (2-DE) computer analysis software packages have existed ever since 2-DE technology was developed, it is only now that the hardware and software technology allows large-scale studies to be performed on low-cost personal computers or workstations, and that setting up a 2-DE computer analysis system in a small laboratory is no longer considered a luxury. After a first attempt in the seventies and early eighties to develop 2-DE analysis software systems on hardware that had poor or even no graphical capabilities, followed in the late eighties by a wave of innovative software developments that were possible thanks to new graphical interface standards such as XWindows, a third generation of 2-DE analysis software packages has now come to maturity. It can be run on a variety of low-cost, general-purpose personal computers, thus making the purchase of a 2-DE analysis system easily attainable for even the smallest laboratory that is involved in proteome research. Melanie II 2-D PAGE, developed at the University Hospital of Geneva, is such a third-generation software system for 2-DE analysis. Based on unique image processing algorithms, this user-friendly object-oriented software package runs on multiple platforms, including Unix, MS-Windows 95 and NT, and Power Macintosh. It provides efficient spot detection and quantitation, state-of-the-art image comparison, statistical data analysis facilities, and is Internet-ready. Linked to proteome databases such as those available on the World Wide Web, it represents a valuable tool for the "Virtual Lab" of the post-genome area.

A simple and inexpensive methacrylate rehydration chamber was built to accommodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 microL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.

The translationally controlled tumor protein (TCTP) is a growth-related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two-dimensional gel electrophoresis, monoclonal antibodies, and 45Ca-gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post-translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.

Several two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases have been established and updated for more than 15 years. Only recently have developments of computer networks and high-speed transfer protocols provided the required tools for sharing comprehensive and hypermedia 2-D PAGE databases. This publication describes the SWISS-2DPAGE database structure. Proteins present in samples of human tissue, cells, cell lines and body fluids are assembled and described in an accessible uniform format. SWISS-2DPAGE can be freely accessed through the World-Wide Web (WWW) network on the ExPASy molecular biology server.

SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), created and maintained at the University Hospital of Geneva in collaboration with the Department of Medical Biochemistry of Geneva University. The proteins have been identified on various 2-D PAGE reference maps by microsequencing, immunoblotting, gel comparison and amino acid composition.

We describe some simple modifications to the micropreparative two-dimensional (2-D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate-electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accommodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N-terminal sequencing is facilitated by the large protein load (1-15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2-D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.

A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two-dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10-25 degrees C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.

The focusing positions in narrow range immobilized pH gradients of 29 polypeptides of known amino acid sequence were determined under denaturing conditions. The isoelectric points of the proteins calculated from their amino acid sequences matched with good accuracy the experimentally determined pI values. We show the advantages of being able to predict the position of a protein of known structure within a two-dimensional gel.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.

N-Terminal sequence analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans-Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R-250 and Ponceau S, we have obtained, in comparison with the non-dried membranes, an enormous increase in the number of detectable proteins.