Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.     

Improving the detection of proteins after transfer to polyvinylidene difluoride membranes.

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.     

Densitometric measurements in a severe light-scattering system

A useful model was needed to study the effects of a light-scattering matrix on densitometric scanning. Such perturbations are encountered in evaluating thin-layer chromatograms and uncleared cellulose acetate electrophoretograms. It is also anticipated that these findings may be extrapolated to all spectrophotometric determinations involving extreme turbidity. Protein separation by cellulose acetate electrophoresis was therefore selected to provide this light-scattering medium. It is an ideal model in that it can be readily used both clear and opaque thus providing two identical systems except for the presence of background opacity (turbidity). Furthermore, by selecting stains whose absorption spectra spanned a broad range it was possible to study the perturbations throughout the visible spectrum. The stains used were Amido black, Coomassie Brilliant Blue, Ponceau S, and bathocuproine sulfonate-copper.     

Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate-electrophoresis method and rapid two-dimensional electrophoresis

Two methods suitable for routine clinical analyses of urinary proteins are presented and compared. The first is a horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique, suitable for simultaneous analysis of 20 native urinary samples. This method uses polyacrylamide gradient gels, prepared with a laboratory-built gel casting device. The second method is a rapid two-dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate-electrophoresis. The first step uses a routine system (Chemetron), the second separation step followed by staining with Coomassie Brilliant Blue R is performed on the PhastSystem. The resulting two-dimensional patterns reveal urinary proteins distributed according to the 5-zone pattern of native proteins (albumin, alpha-1, alpha-2, beta, gamma-globulin) as well as to the logarithm of their molecular weights. Examples of (routine) diagnoses with a special interest in the monitoring of kidney transplant patients are shown.     

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

Gel electrophoretic analysis of chitosan hydrolysis products.

Enzymatic hydrolysis of commercial crustacean chitosan by barley chitosanases was analyzed by subjecting chitosan to electrophoresis in a 10% w/v polyacrylamide slab gel in the presence of 7 M urea and 5.5% v/v acetic acid. Chitosan migrated as a polycation. Chitosan was stained with Coomassie Brilliant Blue R-250 or visualized by ultraviolet transillumination after staining with Calcofluor White M2R. Some chitosan molecules were retarded by gel electrophoresis while small chitosan molecules migrated at the bottom of a 10% w/v polyacrylamide gel. Such analysis revealed that 96 h were necessary to convert all chitosan to oligosaccharides under our assay conditions. Chitosan oligosaccharides generated by enzymatic or chemical hydrolysis were further analyzed by electrophoresis in a 33% w/v polyacrylamide gel containing urea and acetic acid. Coomassie Brilliant Blue R-250 was found to be better than Calcofluor White M2R for staining chitosan oligosaccharides. Chitosan oligomers of four residues (tetramers) or more were easily resolved in such a polyacrylamide gel system. To our knowledge, this is the first report of a gel electrophoretic separation of chitosan and its oligosaccharides.     

Improved detection of human pancreatic proteins separated by two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis using a combined staining procedure.

In order to assess secretory pancreatic proteins in a two-dimensional isoelectric focusing/sodium dodecyl sulphate electrophoresis gel, a highly sensitive double-staining method with Coomassie Brilliant Blue followed by silver stain was used. This combined procedure afforded more distinct spots and additional bands, particularly glycoproteins, than either silver or Coomassie Blue staining alone. As measurements of dye volumes by densitometry have shown, double staining of two-dimensional separated pancreatic proteins is up to twenty times more sensitive than the usual Coomassie Brilliant Blue staining.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.     

Isotachophoresis of proteins

The analytical separation of proteins by isotachophoresis (ITP) was achieved in a short electrophoretic path and with a resolution comparable to that of isoelectric focusing by the appropriate selection of (1) a mixture of ampholytes as spacers to generate linear gradients of electrophoretic mobility and (2) the counter ions chosen to buffer the complete pH gradient generated. This ITP technique is exemplified by the analysis of plasma proteins in agarose gels. Up to 46 samples in the same gel plate were analysed. The resolution was such that at least 30 clear and discrete bands per sample could be observed after staining with Coomassie Brilliant Blue. The resolving power of ITP could be further increased for the study of a particular protein or zone by the selection of suitable spacers and counter ions.     

Recovery of protein by coomassie brilliant blue precipitation prior to electrophoresis.

The interaction of protein with Coomassie Brilliant Blue G-250 results in formation of an insoluble protein-dye complex which can be recovered by centrifugation and redissolved for electrophoretic analysis. The precipitated protein can be washed in acetone to remove excess dye in order to enhance resolution. The residual dye becomes dissociated from the proteins on electrophoresis and can be exploited as a "dye front". The method allows simultaneous protein assay and recovery of microgram amounts of protein from dilute solution and could be widely applied for conserving, concentrating and desalting minute amounts of valuable sample prior to electrophoretic analysis.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.     

Rapid analysis of a peptide by fast-atom bombardment mass spectrometry after polyacrylamide gel electrophoresis

Gel electrophoresis has been a powerful technique for the separation of peptides and proteins for many years. After electrophoresis separation on a polyacrylamide gel, the peptide bradykinin was localized using Coomassie Blue as a staining dye. Excess dye was removed by washing the gel with water. For mass spectrometric analysis, bands containing the peptide were crushed, extracted with acetic acid and the eluent applied to the fast-atom bombardment probe. Under these conditions the protonated molecule of bradykinin was clearly observed. Also apparent were sequence ions at about the same intensity observed from authentic bradykinin.     

Chemical modification of proteins to improve the accuracy of their relative molecular mass determination by electrophoresis

We studied the electrophoretic behavior of basic proteins (cytochrome c and histone III) and developed a carbamylation method that normalizes their electrophoretic size separation and improves the accuracy of their relative molecular mass determined electrophoretically. In capillary zone electrophoresis with cationic hitchhiking, native cytochrome c does not sufficiently bind cationic surfactants due to electrostatic repulsion between the basic protein and cationic surfactant. Carbamylation suppresses the strong positive charge of the basic proteins and results in more accurate relative molecular masses.     

Polyelectrolyte complexes. V. Solid‐state properties of some polycation/azo dye complexes controlled by the dye structure

The solid‐state properties of some polycation/azo dye complexes according to the dye structure were studied in this work. One polycation contained about 95 mol % N,N‐dimethyl‐2‐hydroxypropyleneammonium chloride units in the backbone (PCA₅), and eight azo dyes, different in either the number of sulfonic groups or their distribution, were used as opposite components. The selected azo dyes were as Crystal Scarlet, Congo Red, Crocein Scarlet MOO, Ponceau SS, Amaranth, Ponceau S, Direct Blue 1, and Direct Red 80. Information on the compensation degree of the oppositely charges was obtained by the elemental analysis of the solid‐state polycation/dye complexes (the experimental contents of chlorine, nitrogen, and sulfur were compared with the calculated values). Differential scanning calorimetry was employed to probe the strength of the intermolecular interactions in the PCA₅/dye complexes. Wide‐angle X‐ray diffraction was used to assess the supramolecular order of the solid‐state complexes. The physical properties of the PCA₅/azo dye complexes (the complex stoichiometry, glass‐transition temperature, decomposition temperature, and degree of supramolecular order) were influenced mainly by the dye structure but also by the polycation concentration and the presence of NaCl. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 264–272, 2003     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins

2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Transformation of textile dyes by white-rot fungus Trametes versicolor

We have investigated transformation of eight industrial dyes by a whiterot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red, Reactive Violet 5, Reactive Blue 28, and Ponceau Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H₂O₂-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced by Mn²⁺ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor.