Focusing In pH Gradients Research Articles

English

Medicago truncatula leaves were used as the experimental materials. Total proteins of leaves were extracted by trichloracetic acid (TCA)-acetone method and proteins had a better separation using gel strips, forming an immobilized non-linear 3 to 10 pH gradient focusing 123,000 vhr combined with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie Brilliant Blue G-250 and digitalized gels were analyzed using the PDquest 8.0.1 software. The results indicated that 931 protein dots were detected in the gel. A technology suitable for the M. truncatula leaves protein extraction by TCA/acetone and a protocol for two-dimensional electrophoresis (2-DE) was established, which provides technical support for M. truncatula leaf proteome research. Key words: Medicago truncatula, proteome, two-dimensional polyacrylamide gel electrophoresis (2-DE), isoelectrofocusing (IEF).

Recent developments in preparative free flow isoelectric focusing.

Continuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow- or broad-range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.

Comparison of Milk Proteins Using Preparative Isoelectric Focusing Followed by Polyacrylamide Gel Electrophoresis

The major proteins in milks from bovine, caprine, porcine, and murine animals and from humans were compared using a two-dimensional analysis method. In the first dimension, proteins were separated by their isoelectric points using preparative isoelectric focusing in pH gradient of 3 to 10. Twenty fractions from each sample were then analyzed by urea-PAGE and SDS-PAGE. Two-dimensional gels showed characteristic patterns for each milk. Major bovine milk proteins were identified and used as reference for proteins of other mammals. Additionally, some peptides resulting from plasmin hydrolysis were characterized. Caprine milk proteins showed a pattern similar to that of bovine milk except for the absence of αs1-caseins. α-Lactalbumin of bovine and caprine milks resolved as two bands in an immunoblot using bovine α-lactalbumin antibody. Each band corresponded to normal and glycosylated α-lactalbumin. Human, porcine, and murine milk proteins were totally different from those of ruminant milks on the two-dimensional gels. Two-dimensional analysis using preparative isoelectric focusing, followed by PAGE, was a useful method to compare major milk proteins in several mammals because of the rapid simultaneous separation into 20 fractions. This fractionation allows additional analytical procedures for more efficient comparison of chemical and physical properties of the proteins.

Comparison of the polypeptide composition of cystic fibrosis plasma with normal plasma by high resolution electrophoresis

Cystic fibrosis and normal plasma proteins were compared by the method of high resolution two-dimensional electrophoresis in polyacrylamide gels. For the first dimension, samples were treated with urea and dithiothreitol and then subjected to isoelectric focusing in pH gradients of 3.5–10.0 or 5.0–8.0. The second dimension involved sodium dodecyl sulfate electrophoresis into gels of 10–20% polyacrylamide gradients. In one “blind” experiment, an attempt to segregate 12 plasma samples into six cystic fibrosis and six normals based on the polypeptide patterns was unsuccessful. Experiments using known cystic fibrotic and normal plasma samples (three further samples of each), depleted of albumin prior to electrophoresis, also failed to confirm the presence of cystic fibrosis related proteins or peptides. We have not been able to substantiate, by electrophoretic means, changes of sialic acid content of any plasma glycoprotein which might be expected to occur, for example, as a result of the postulated altered sialyltransferase activity in cystic fibrosis liver.

Isoelectric focusing in continuous-flow electrophoresis: I. Separation of mixed red blood cells of different species

A noncommercial continuous-flow isoelectric focusing (CIF) apparatus which was formerly applied to separate mixtures of proteins was used to study the separation of red blood cells (RBC's) of different species. A mixture of human, mouse and rabbit erythrocytes, a good model for demonstration of cell separation by CIF, was completely separated into the three components. The separation was performed by isoelectric focusing in pH gradients, 3–10 and 5–7, using Ampholine carrier ampholytes at a field strength of 110 V/cm and a flow-through time of RBC's of 7 min. The isoionic points of human RBC's both determined by CIF and calculated from electrophoretic mobility measurements by extrapolation to zero electrophoretic mobility and zero ionic strength were found at pH 5.6–5.7. The method of CIF which is presently used to isolate a pure lysosomal fraction seems to be a valuable method for the separation of mixtures of cells or cell organelles.

Evidence for the formation of methoxyl groups of ferulic and sinapic acids in Bambusa by the same O-methyltransferase

The two methylation reactions, i.e. caffeate to ferulate (FA) and 5-hydroxyferulate to sinapate (SA), in the biosynthesis of guaiacyl and syringyl lignins in angiosperms were demonstrated to be catalyzed by the same enzyme in bamboo. This follows from the facts that: the ratio (SA/FA) obtained for O-methyltransferase remains constant during purification of the enzyme; chromatography on DEAE-cellulose, Sephadex G100 and G200, and analyses by polyacrylamide gel electrophoresis and isoelectric focusing in pH gradients showed that the two methylating activities belonged to a single enzyme protein; caffeate and 5-hydroxyferulate compete each other in the formation of the enzyme-substrate complex, the latter substrate showing greater affinity for the enzyme. Thus, feedback control may operate at the methylation step, caffeate to FA, in biosynthesis of angiosperm lignin.

Isoelectric focusing in free Ampholine solution and attempts at isoelectric focusing in pH gradients created in ordinary buffers.

Isoelectric focusing in free Ampholine solution and attempts at isoelectric focusing in pH gradients created in ordinary buffers.

Microelectrofocusing of proteins in capillary gels

A microtechnique of isoelectric focusing in acrylamide is described which allows the separation of proteins in the millimicrogram range. Isoelectric focusing in pH-gradients formed by aliphatic aminocarboxylic acids (1,2) has recently become a widely used method for the separation of proteins (3). For the analysis of microgram quantities of material, isoelectric focusing in gel columns of the type used for disc electrophoresis, has been shown to be a very efficient means (4–8). Microelectrophoresis in capillary columns (9) has earlier been applied to the separation of proteins in the millimicrogram range (10–12). By use of the equipment devised for microelectrophoresis, it was possible to develop a technique for the electrofocusing of proteins on the same scale. While electrophoresis in capillaries deserves special precautions and manipulations to achieve sharp separation of the sample constituents (9, 12), microelectrofocusing is very easily performed because sharp zones are automatically formed during the run.

Isoelectric focusing in pH gradients--a technique for fractionation and characterization of ampholytes.

This chapter contains sections titled: Introduction History The Natural pH-Gradient Stabilization Against Convection Density Gradient Electrofocusing Supplementary Instructions for Sucrose Density Electrofocusing Gel Electrofocusing Zone Convection Electrofocusing Conclusions

Studies on extracellular proteins from Staphylococcus aureus. IV. Separation of α-toxin by isoelectric focusing

Three strains of Staphylococcus aureus (V8, M18, and Wood 46) were grown in batch and continuous culture in ‘CCY medium’. Hundred-fold differences in toxin production were found for strain V8 and Wood 46 when the bacteria were grown to a density of 6–8 g/l (dry weight). Dialyzed culture supernatants and crude concentrated toxin preparations were subjected to isoelectric focusing in pH gradients. The α-toxin turned out to be separable into three or four components with different isoelectric points (p I's). The main component, α Ia, was found to contain roughly 80% of the total haemolytic activity and had a p I of 8.5. The carrier ampholytes and the sucrose had a stabilizing effect on the toxins. Yields of 70–140% were calculated. The degree of purification and the specific activities are estimated. Electrofocusing experiments were performed in 6 M urea and gave analogous results which was also the case for ‘heat-purified α-toxin’. After Sephadex G-75 chromatography one single peak of α-toxic activity was found. Finally, isoelectric focusing of single fractions of α Ia IIIa showed an interconversion phenomenon. These studies suggest that the multiple forms of α-toxin differ in conformation.

Studies on extracellular proteins from Staphylococcus aureus

Abstract Five extracellular deoxyribonucleases produced during growth of Staphylococcus aureus strains V8, Wood 46 and M 18 have been separated by isoelectric focusing in pH gradients. The same two major components (deoxyribonucleases A and B) and three minor ones (deoxyribonucleases C, D and E) were obtained through ‘high-resolution focusing’ from all three strains. Their isoelectric points were estimated at 4° and the five components were found to be isoelectric: deoxyribonuclease A, pH 2–3; deoxyribonuclease B, pH 10.1; deoxyribonuclease C, pH 4.5–5.5; deoxyribonuclease D, pH 6.0–6.5; and deoxyribonuclease E, pH 8.2–8.7. Such physicochemical characteristics as ionic requirements, effect of pH and ionic strength upon enzyme activity, were studied along with investigations of heat stability and degradation of native and denatured DNA. A micrococcal nuclease prepared according to Anfinsen 8 also was subjected to isoelectric focusing and found to have an isoelectric point of 10.1 thus corresponding to the most alkaline deoxyribonuclease (deoxyribonuclease B) found in this investigation.