Carrier Ampholyte Isoelectric Focusing Research Articles

Generalized Physicochemical Model for the Natural pH Gradient in Classic IEF

In this paper we present a generalized physicochemical model for carrier ampholyte isoelectric focusing techniques. Accurate analysis of electrokinetic phenomena can only be performed when the physicochemical interactions of the carrier matrix with the electrode solutions are taken into consideration. It should also be taken into account that the majority of the constituents of electrolyte systems are weak, multivalent acids and bases, which have complex stoichiometric behavior, intricately linked with local transport phenomena. A generalized formulation to couple the migration/diffusion of weak electrolytes with transport phenomena is presented along with a broad treatment of macroscopic transport, which leads ultimately to the creation of an ampholyte pH gradient. These formulations and a novel scheme for the mechanism of mass transport constitute the core of the present paper.

Carrier Ampholyte Isoelectric Focusing Based Two-Dimensional Electrophoresis in Preliminary Screening of Differential Proteomics Analysis

Two-dimensional electrophoresis is a current method for separating complex protein mixtures of a given sample in different states. In this study an improved carrier ampholyte isoelectric focusing method has been evaluated for its capacity for preliminary screening of expressional proteomics subjects. In comparison with current carrier ampholyte isoelectric focusing, this method showed enough resolution power to display major expressional changes in proteomic samples and demonstrated it can be used as a substitution for the immobiline based isoelectric focusing method.

New approach to calculating and predicting the ionic strength generated during carrier ampholyte isoelectric focusing

A serious drawback of the carrier ampholyte isoelectric focusing is the undetermined ionic strength at which the proteins separate. We tried to study its effect in order to bridge the gap between theory and practice giving a quantitative and qualitative description. Using certain electrode systems, we managed to calculate the discrete values of the ionic strength in a random position between the electrodes. As a rule, higher values of up to 10 −3 mol/l were obtained near the electrodes, which gradually decreased towards the middle of the carrier reaching 10 −7 mol/l. A relationship between the measured isoelectric points (p I) of some proteins and the ionic strength was found. In the present paper, we report our findings on the dynamics of the process referring to the origin of ionic strength, its formation and alteration during the isoelectric focusing process.

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis.

A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.

Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells.

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Discrimination Between Common and Necrotic Strains of Potato Virus Y by Denaturing Isoelectric Focusing

Purified virions from seven isolates of the common strain (PVY O ) and six isolates of the tobacco veinal necrotic strain (PVY N ) of potato virus Y were examined by denaturing carrier-ampholyte isoelectric focusing (CA-IEF). All gave a similar pattern of one major and either two or three minor bands. The pattern of all PVY N isolates was identical and the coat protein was acidic (pI=6.1 in all cases), whereas the PVY O isolates could be divided into three distinct groups, all of which were more alkaline (pI=6.8, 7.2, and 7.4). Isoelectric focusing followed by electroblotting and immunodetection showed the bands to be of viral origin. SDS-PAGE gave a single major band of 29.5 kDa in all cases. Isolation of virions from either potato or tobacco gave the same results

Immobilised pH gradient isoelectric focusing of wool proteins.

Conventional carrier ampholyte-isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient-isoelectric focusing (IPG-IEF) for wool proteins, S-amidomethyl wool proteins were analysed using IPG-IEF. Over twenty bands were visible on a pH 4-7 IPG-IEF gel. IPG-IEF was combined with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG-IEF appears to be ideally suited to the separation of wool proteins. The resolution of two-dimensional electrophoresis using IPG-IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.

Additives for immobilized pH gradient two-dimensional separation of particulate material: Comparison between commercial and new synthetic detergents

We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commerical compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3–12, the concentration of urea compatible with 2% detergent increases from 4.5 m for the parent molecule up to 7 m. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl- N,N-dimethylammonio-3-propanesulfonate of a major protein (p I = ca. 5.5, M r = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a p K of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing. In its solubilizing properties, A15 is similar to Chaps: at a 2% concentration it is compatible with 10 m urea solutions.

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the p I. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.

Isoelectric focusing and non‐isoelectric precipitation of ferritin in immobilized pH gradients: An improved protocol overcoming protein‐matrix interactions

AbstractIt has been impossible, up to now, to focus ferritin (a large, ca. 450 000 Da, ironstorage protein ubiquitous in mammalian systems) in immobilized pH gradients (IPG). This was not a sieving problem, as ferritin focuses readily in a 4%T polyacrylamide (or polytrisacryl) gel in conventional carrier ampholyte isoelectric focusing. A strong interaction with the IPG matrix was suspected, but an auto‐buffered (pH 6.0) soluble IPG polymer failed to precipitate ferritin from solution, although it altered its visible spectrum, suggesting the formation of soluble complexes. By a Takeo plot, the dissociation constant (Kd)of the ferritin‐IPG matrix complex was estimated to be 0.1 mM. However, even a 100‐fold dilution of Immobilines into the IPG matrix with a concomitant addition of competing ions (up to 3 % carrier ampholytes) failed to produce good focusing patterns. An unsuspected, hitherto un‐reported, precipitating power of free Immobilines in solution on the ferritin macroion was found to be the key to the riddle. The precipitating ions are (in order of aggregation power): pK 9.3 > pK 7.0 > pK 6.2. None of the acidic Immobilines (pKs 3.6, 4.4 and 4.6) and, among the basic, only the pK 8.5, exhibit any precipitation power on ferritin and other complex protein mixtures. In a new IPG pH 4–5 interval, containing only the pK 4.6 as buffering and the pK 8.5 Immobilines as titrant ions, excellent focusing patterns of ferritin were obtained. It is hypothesized that the precipitating Immobilines act as cationic detergents, and are able to cross‐link macroions with a mixed‐type, hydrophobic‐ionic interaction at the two extremities of the molecule. Guidelines are given for handling this major problem on a short‐term basis, the longterm goal being the replacement of the ‘unfavorable’ Immobilines (pKs 9.3, 7.0 and 6.2) by new chemical structures of the desirable type (the non‐precipitating pK 8.5 species).