Correlation between native and desialyzed forms of orosomucoid

The correlation between ORM phenotypes in untreated serum and ORM phenotypes after isoelectric focusing of neuraminidase-treated serum is demonstrated. ORM subtypes were determined using isoelectric focusing in immobilized pH gradients. Population genetic studies of ORM polymorphism in the Galician population were also carried out.     

Isoelectric focusing of cells using zwitterionic buffers.

A simple method of isoelectric focusing of cells is described. The pH gradient, superimposed on a density gradient, is developed by generating opposing concentration gradients of two zwitterionic buffers. The method can be used as a cell separation technique or as a means of characterizing the cell type on the basis of the focusing pH. Focusing is rapid and thus the method is of special advantage in its application to cells.     

A new α1-antitrypsin allele PI*Poki: Isoelectric focusing with immobilized pH gradients as a tool for identification of PI variants

Summary A new rare genetic variant PI Poki in the protease inhibitor system found in two Japanese subjects, was compared with some PI P variants using several electrophoresis systems. Although this variant was slightly anodal to P by separator isoelectric focusing, it could be readily distinguished by isoelectric focusing with immobilized pH gradients: the mobility of Poki became cathodal to P. This method proved to be a powerful tool for identification of PI variants.     

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.     

Scanning isoelectric focusing in small density-gradient columns: I. Use of a Standard Spectrophotometer Cuvette for Focusing. Chemical Modification of Proteins by Migrating Reactive Ions

By the method of isoelectric focusing in a sucrose density gradient, small protein samples (less than 100 μg) have been separated and analyzed within 2 hr, using as electrolysis column a commercial standard quartz spectrophotometer cuvette, equipped with platinum electrodes and placed in an optical scanning device. Preparation of the cuvette prior to an isoelectric focusing experiment required about half an hour with no external apparatus, as the density gradient was created spontaneously in the cell by free interdiffusion of sucrose solutions. The cuvette temperature could be controlled by circulating water. The optical detection device permitted repeated scanning of the cuvette during the electrolysis process, thus providing information about the events occurring to a protein during focusing or prolonged electrolysis. By scanning with wavelength at the positions where the proteins have focused, their absorption spectra were obtained. the isoelectric points of separated proteins were estimated by fractionation of the cell contents and subsequent pH measurements on the fractions.The present paper also describes how individual Ampholine components, or groups of components, in their focused states gave rise to easily detected refractive-index gradients within the cell. The optical scanning device has been built in such a way that interference of these gradients with absorption measurements was abolished.Application of the technique to the isoelectric separation of commercial sperm whale myoglobin is reported. Ferrous or ferric forms of the focused myoglobin components were obtained by migration of reducing or oxidizing agents through the zones.     

An apparatus for immunoelectrofocusing. A study of squid tyrosinase.

An apparatus which facilitates immunoelectrofocusing is described. Polyacrylamide is used as the support medium for both the thin-layer isoelectric focusing and immunodiffusion stages. The pH gradients formed during electrofocusing are reproducible and sufficiently stable. Immunoelectrofocused and isoelectric focused patterns indicate that at least one inactive form and four active forms of squid tyrosinase exist.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.     

Isoelectric focusing in immobilized pH gradients: Principle,methodology and some applications

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described.Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacitu; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strenght.     

Isoelectric focusing in polyacrylamide gel using buffer electrode solutions.

Isoelectric focusing in ampholytes of pH 6–8 range has been carried out in polyacrylamide gels using ammonia buffer at pH 10.0 and acetate buffer at pH 4.0 for the cathode and anode solutions, respectively. This system requires low voltages but compares well with isoelectric focusing using strong acid and strong base electrode solutions. The advantages of this method are the less extreme pH's in the electrode solutions and lower resistance in the neutral region of the pH gradients.     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

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.     

Isoelectric focusing of boar spermatozoa.

The isoelectric points of washed spermatozoa from intact boars and from boars after removal of the seminal vesicles were determined using isoelectric focusing on natural pH gradients. Normal boar spermatozoa focused at a higher pH than spermatozoa from boars without seminal vesicles. The isoelectric point of the latter was increased to a value approaching normal by preincubation in normal seminal plasma. This indicates that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Nonisoelectric focusing in buffers.

Stable pH gradients were formed and focusing of proteins was carried out in polyacrylamide gels containing mixtures of simple, amphoteric buffers, replacing the Ampholine hitherto used in isoelectric focusing (IF). Stable pH gradients can also be formed between acid anolyte and basic catholyte if Ampholine is replaced by nonamphoteric buffers. The fact that focusing can be carried out with nonampholytes shows that focusing in this case is, and in all other cases may be, nonisoelectric. It is postulated that the pH gradient in IF forms by steady-state stacking (isotachophoresis) and forms within the stack. In distinction to ordinary steady-state stacking, however, the stack remains confined within the gel (or density gradient) since the strong acid and base in the electrolyte reservoirs bar by deprotonation or electrostatic repulsion migration into the electrode chambers.     

Isoelectric focusing and isoelectric points of bovine liver histones

A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of histones.     

Isoelectric focusing of carboxypeptidase N.

Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing.     

Isoelectric focusing in Pevikon slabs

A mixture of Pevikon C-870 and Sephadex G-75 superfine is proposed for isoelectric focusing in granular beds. The beds are easy to prepare, and pH gradients are stable for 36 hr under the given conditions. Precise and clean fractionations are obtained with excellent recoveries of focused substances.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.     

箭毒木种子蛋白质样品制备及双向电泳改良方法

建立箭毒木（Antiaris toxicaria）种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris—HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris—HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris—HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。