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C Gelfi

Publications and source records attributed to C Gelfi.

105 records · Page 6Linked to original sources
PubMed1986

pH measurements in ultranarrow immobilized pH gradients.

It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 microliter of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 microliter) are eluted in 900 microliter of 100 mM KCl and the pH assessed with a differential pH meter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert delta pH values into an actual pH curve. The reproducibility of the system is better than +/- 0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2-3 pH unit) carrier ampholyte interval into a narrow (0.2-0.3 pH unit) one.

Hydrogen-Ion Concentration↗
PubMed1986

Direct recovery of proteins into a free-liquid phase after preparative isoelectric focusing in immobilized pH gradients.

A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The transfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: short electrophoretic times; avoidance of anodic oxidation; chilling of the buffer in the anodic chamber; and use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).

Enzymes↗
PubMed1984

Protein C antigen is not an acute phase reactant and is often high in ischemic heart disease and diabetes.

Protein C, an antithrombotic protein, was measured immunologically in 299 patients with clinical conditions associated with a high frequency of venous or arterial thromboembolism. The mean protein C antigen (PC:Ag) level was high for 48 patients with ischemic heart disease and, to a lesser extent, for 95 diabetics. In 28 patients with thrombotic strokes, 48 patients with proximal deep-vein thrombosis and in 80 patients with localized or metastatic tumors, mean PC:Ag was normal. Comparison of the pattern of changes of PC:Ag levels with those of fibrinogen, orosomucoid and prothrombin in 21 patients during the postoperative period and in 20 patients with active rheumatoid arthritis ruled out the possibility that high PC:Ag is non-specific, acute-phase reaction to inflammation, tissue injury or neoplastic growth. Therefore, high PC:Ag might be specifically related to the thrombotic tendency of these patients, but the mechanism of such a relationship remains to be clarified.

Adolescent↗
PubMed1984

Fractionation techniques in a hydro-organic environment. II. Acryloyl-morpholine polymers as a matrix for electrophoresis in hydro-organic solvents.

The properties of gels prepared either from acryloyl-morpholine (ACM) or from its mixtures with acrylamide and crosslinked either with bisacrylylpiperazine or with methylenebisacrylamide have been described. ACM-containing gels are compatible with organic solvents. If polymerized in water and dried, they are able to reswell, e.g., in dimethyl sulfoxide or dimethylformamide. If polymerized in presence of dimethylformamide, they form perfectly clear gels, whose mechanical properties are by far superior than those of similar plain polyacrylamide formulations.

Acrylic Resins↗
PubMed1984

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels.

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-155, 157-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to be carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37 degrees C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

Acrylic Resins↗
PubMed1983

Preparative isoelectric focusing in immobilized pH gradients. II. A case report.

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.

Computers↗
PubMed1980

Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation.

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.

Alcohol Oxidoreductases↗
PubMed1979

Isoelectric focusing of oligopeptides: detection by specific stains.

By using ultrathin (350 micrometers) polyacrylamide gels, which at the end of the fractionation are pasted to filter paper and dried in an oven at 110 degrees C, and after isoelectric focusing it has been possible to detect oligopeptides in the di- to tetradecapeptide range, which could not be detected by protein staining techniques. This is achieved by developing a series of specific stains for the following amino acids: Arg, Tyr, His, Trp, Met and Cys. Except for Met and Cys, the detection limits appear to be in the order of 0.2--2 micrograms of free amino acid loaded in the gel. The Pauli reaction for His and Tyr and the Sakaguchi stain for Arg can be developed sequentially in the same gel, thus allowing the detection of four different amino acids since, under these conditions, also Trp reacts. Unfortunately, more general reactions, such as the permanganate, the 'Lowry' and the ninhydrin stains, cannot be utilized since the carrier ampholytes react very strongly with all these reagents.

Amino Acids↗
PubMedDate not supplied

Photopolymerization of polyacrylamide gels with methylene blue.

Photopolymerization of polyacrylamide gels in the presence of methylene blue (100 microM) and a redox couple (1 mM sodium toluenesulfinate, a reducer, and 50 microM diphenyliodonium chloride, an oxidizer) has been investigated. The gel point, i.e. the time needed for onset of gelation upon illumination, has been found to lengthen progressively at lower temperatures and at lower light intensities. If the three catalysts are progressively diluted, the gel point does not vary for a threefold dilution, but gelation is greatly hampered below a 1:5 dilution of the three effectors. Photobleaching has been assessed as a function of liquid layer thickness (from 0.5 to 2 mm), of a progressive dye dilution (down to a fourfold dilution) and as a function of temperature. A maximum of elastic modulus is located in correspondence to a minimum of permeability (both situated at 5% cross-linker). It is found that methylene blue-activated polymerization produces polyacrylamide gels with elastic properties which are higher than in persulfate-activated gels, so far the most popular matrices for electrokinetic separations. Due to the ease of preparation, the full control of all experimental parameters, and the lack of oxidizing power of this catalyst system (as opposed to the strong oxidation power of persulfate catalysis), methylene blue catalysis is advocated as a valid alternative to other redox systems.

Acrylic Resins↗
PubMedDate not supplied

Casting immobilized pH gradients into cylindrical polyacrylamide gels.

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).

Acrylic Resins↗
PubMedDate not supplied

Immunoblotting from immobilized pH gradients. The case of alpha 2-macroglobulin.

Guidelines for effective blotting of proteins from immobilized pH gradients with a soft polyacrylamide matrix (e.g. T% = 3) include: thick (1 mm) IPG slabs, electrotransfer in a buffer tank in the presence of 0.1% SDS, nitrocellulose of the sturdiest type, thorough removal of all IPG fragments before further processing of the membrane. For alpha 2-M, IPG on a 4-6.5 gradient followed by enzyme-linked immunodetection allows the recognition of a complex pattern with several bands centered around pI 5.1. The procedure may also reveal the desialylated forms of alpha 2-M (microheterogeneity reduced to 2-3 bands), the native subunits (after reduction with thiols) and the denatured half molecules (in the presence of 8 M urea).

Hydrogen-Ion Concentration↗
PubMedDate not supplied

Capillary electrophoresis of DNA for molecular diagnostics: an update.

The use of capillary electrophoresis in sieving liquid polymers for DNA analysis in the biomedical field is reviewed. One hundred and six references have been compiled and divided into the following categories: 1) screening for inherited human genetic defects, 2) quantitative gene dosage, 3) microbiology/virology, 4) forensic analysis, and 5) therapeutic DNA (antisense nucleotides).

DNA↗
PubMedDate not supplied

Capillary isoelectric focusing and isoelectric buffers: an evolving scenario.

The present review offers a new look at capillary isoelectric focusing (cIEF) by centering on the most troublesome aspects of the technique, namely: 1) how to modulate the slope of the pH gradient, for increasing resolution (equivalent to pH gradient engineering, as easily available in immobilized pH gradients); and 2) how to keep proteins in solution at (and in the proximity of) the pl value. A simple solution is offered in the first case: addition, to the standard 2-pH-units interval, of separators or spacers, i.e., of amphoteric molecules (either single or in combination) able to locally flatten the pH and increment resolution. Examples of the separation of fetal and glycated hemoglobins are provided. In the second case, a unique solubilization power (while maintaining full protein integrity and enzyme activity) is obtained if class I solubilizers are used. They consist of mixtures of sugars (e.g., sucrose and sorbitol) at ca. 1 M concentration, with zwitterions (up to 1 M) such as the class of nondetergent sulfobetaines, but also taurine and some of the Good's buffers (e.g., CAPS). In these solvents, the protein exists in a state of superhydration and its solubility is greatly augmented. The review ends with an excursus on the use of isoelectric buffers in zone electrophoretic separations. Such isoelectric buffers offer unique advantages: They permit very-high-voltage gradients (up to 1000 V/cm) and thus minimize analysis times (down to a few min in 30-35 cm long capillaries). This results in a marked increase in resolution, due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of oligonucleotides.

Animals↗