Electrophoresis

Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS‐MODEL, a server for automated comparative protein modeling and of the SWISS‐PdbViewer, a sequence to structure workbench. The Swiss‐PdbViewer not only acts as a client for SWISS‐MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS‐MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.

A new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one‐and two‐dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non‐structural, tobacco mosaic virus‐specific 126 kDa protein directly in the one‐dimensional protein pattern of infected protoplasts by a staining procedure.

We describe a modification of two‐dimensional (2‐D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2‐D gel, post‐run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2‐D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.

SWISS‐MODEL pioneered the field of automated modeling as the first protein modeling service on the Internet. In combination with the visualization tool Swiss‐PdbViewer, the Internet‐based Workspace and the SWISS‐MODEL Repository, it provides a fully integrated sequence to structure analysis and modeling platform. This computational environment is made freely available to the scientific community with the aim to hide the computational complexity of structural bioinformatics and encourage bench scientists to make use of the ever‐increasing structural information available. Indeed, over the last decade, the availability of structural information has significantly increased for many organisms as a direct consequence of the complementary nature of comparative protein modeling and experimental structure determination. This has a very positive and enabling impact on many different applications in biomedical research as described in this paper.

A modified Neuhoff's colloidal Coomassie Blue G‐250 stain is reported, dubbed “blue silver” on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The “blue silver” exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the “blue silver” exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of bovine serum albumin (BSA) gave a detection limit (signal‐to‐noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of “blue silver” as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two‐dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.

The level of genetic variation revealed by two‐dimensional electrophoresis of proteins from seedlings of two wheat lines strongly depends on the technical procedures. Improvements in extraction and electrophoresis procedures relative to earlier experiments on the same material led to a significant increase in the genetic variation revealed: 15.2 % instead of 6.7 % of the spots were genetically variable. The improved procedure is based on (i) precipipation of proteins from wheat seedlings with trichloroacetic acid and acetone, (ii) solubilization of the proteins with a solution containing urea, potassium carbonate and sodium dodecyl sulfate, (iii) isoelectric focusing in an optimized pH gradient, obtained with a mixture of carrier ampholytes (Pharmalyte and Servalyt), and (iv) running elecrophoresis in the second dimension on gels with increased surface.

The two‐dimensional electrophoresis (2‐DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211–234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007–4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels – these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2‐DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 × 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready‐made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2‐DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2‐DE of complex protein mixtures. The 2‐DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2‐DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus × Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05–0.1 ng protein per band. Total staining time with the new procedure is 75 min.

Here we present a simple and low‐cost production method to generate paper‐based microfluidic devices with wax for portable bioassay. The wax patterning method we introduced here included three different ways: (i) painting with a wax pen, (ii) printing with an inkjet printer followed by painting with a wax pen, (iii) printing by a wax printer directly. The whole process was easy to operate and could be finished within 5–10 min without the use of a clean room, UV lamp, organic solvent, etc. Horse radish peroxidase, BSA and glucose assays were conducted to verify the performance of wax‐patterned paper.

We describe the extraction and enrichment of membrane proteins for separation by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three‐step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris‐base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N‐terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2‐D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2‐D PAGE using broad‐range pH 3.5–10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.