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At least 19 recordsLinked to original sources
PubMed2006

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations.

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3<pI<10 range, were prepared by blending. The master blend served as the feed mixture in binary isoelectric trapping separations that used the buffering membrane to be characterized. The pI value of the most basic UV-absorbing or fluorescent carrier ampholyte collected in the anodic separation compartment, determined by full-column imaging capillary isoelectric focusing analysis, indicates the operational pH value of the separation membrane.

Acrylates↗
PubMed1985

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing.

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Ampholyte Mixtures↗
PubMed1997

[Capillary isoelectric focusing of protein].

A capillary isoelectric focusing (CIEF) method was developed for the separation of proteins with different pI. Uncoated capillary (57 cm x 50 microm i.d.) was used instead of commercial coated column because of its ease in use and a longer lifetime. Polymeric additive, methylcellulose (MC-1500), acts as dynamic coating of the silica wall to suppress electroendosmosis. TEMED was used to extend the pH range of ampholine. Taurine was added to the ampholyte mixture to eliminate the aggregation of proteins. The uniform design, a new experimental design method suited for experiments of a large number of factors and levels, was used for the optimization of experimental conditions, such as concentrations of ampholine, MC-1500, TEMED and Taurine. U6* (6(4)) table was chosen to arrange experiments. Regression equations of maximum current, mean migration time and criterion of resolution power were obtained after the treatment of experimental results by SAS software. Optimized experimental conditions were obtained according to multivariate regression equations. Carrier ampholyte which consists of 3.2%-4.8% ampholine 3-10, 0.06%-0.18% MC-1500, 0.8% TEMED and 1%-2% taurine will produce good resolution. Cathode and anode solutions were 20 mmol/L NaOH and 20 mmol/L HsPO4, respectively. Low pressure (34.5kPa) was applied at the inlet of the capillary simultaneously after 31 minutes of isoelectric focusing at 25 kV to mobilize the focused acidic protein zone to pass through the detector. Human serum albumin (HSA) and human epidermal growth factor (hEGF) were separated by this method using an ampholyte mixture of 4% ampholine 3-10, 0.8% TEMED, 1% taurine and 0.12% MC-1500. Data analysis indicated that the coefficients of variation of pressure and the method are all 1.2%. The results demonstrated that the method established is applicable to basic, neutral and acidic proteins, and can be used for the determination of pI by internal calibration.

Ampholyte Mixtures↗
PubMed1999

Carrier ampholytes rehabilitated: gel isoelectric focusing on pH gradients visualized in real-time by automated fluorescence scanning in the HPGE-1000 apparatus.

All synthetic carrier ampholyte mixtures (SCAMs) contain some naturally fluorescing carrier ampholytes (CAs). The detection of these during isoelectric focusing (IEF), using gel electrophoresis apparatus with intermittent scanning of fluorescence, allows one to follow in real-time the "life cycle" of the pH gradient, i.e., its genesis, steady-state, and decay. The most prominently fluorescing CAs can be calibrated by pH measurement at or after the steady state ("calibration CAs"). By application of the calibration CAs, the fluorescence pattern of CAs can be interpreted in terms of pH gradient (p/s). Simultaneously with the visualization of the pH gradient in that way, protein samples can be detected by "fluorescence reduction" and assigned pH values in dependence on focusing time and, at or after the steady state of the protein, pI' values. The method remedies the inherent blindness of IEF with regard to the state of the pH gradient within its limited "life cycle". It allows one to load the sample at a time when the shape of the pH gradient is optimal for the purpose of its resolution from neighboring components. The visualization of the cathodic drift during IEF eliminates the danger to resolution and to loss of sample associated with "blind" IEF. Most importantly, the possibility to follow the pH in the position of the protein as a function of time provides an objective, accurate measure of the pI' not available from pH measurement at an arbitrary focusing time. The method therefore preempts the advantage of using an IEF method which is free of the pH gradient drift, i.e., immobilized pH gradient (IPG)-IEF. Moreover, it preserves the "natural pH gradient", does not present any of its sample entry problems and those due to very low conductance, and is compatible with agrose gels and their relatively diminished restrictiveness to migration.

Ampholyte Mixtures↗
PubMedDate not supplied

Modeling two-component isoelectric focusing buffers in a vortex-stabilized electrophoresis apparatus.

Defined buffer systems have been considered by researchers as replacements for synthetic carrier ampholyte mixtures in IEF in order to reduce both costs and deleterious protein-ampholyte interactions. In this paper, a mathematical model for the behavior of two-component pH gradients during IEF is applied to a vortex-stabilized electrophoresis apparatus. Equations for mass conservation, molar fluxes, and the electric field describe the behavior of the components in the electric field. Equilibrium constants are used in the model to account for interconversion between the positive, negative, and neutral states for each component. The model was applied to predict pH gradients using two different defined buffer systems. The model's predictions fell within the 95% confidence interval of a least-squares fit to the experimental pH gradients except in the regions near the ends of the pH gradients, where large pH excursions occurred during the experiments.

Ampholyte Mixtures↗
PubMed2004

Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus.

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.

Ampholyte Mixtures↗
PubMed1982

Charge microheterogeneity of the major capsid protein of polyoma virus.

The behavior in isoelectric focusing of the major capsid polypeptide VPI of several strains of polyoma virus was studied. Two previously recognized phenomena were reexamined, namely, (i) the separation of the VP1 polypeptide into multiple subspecies differing only slightly from each other in apparent isoelectric point and (ii) strain differences in the overall apparent net charge of the family of VP1 subspecies. It was found that the pattern of subspecies was reproducible when focusing was initiated from either the basic or acidic region of the gel, keeping the ampholyte mixture constant. However, individual subspecies were unstable, and labeled polypeptide could be shifted dramatically by either refocusing of separated subspecies or by altering the concentration of ampholytes. These findings suggest that protein-protein and protein-ampholyte interactions play an important role in the generation of this charge heterogeneity. The basis for the overall charge difference between the VP1 of 3049 virus and several other strains (lpD, lpS, ts59, and A2) was studied, using recombinant viruses constructed of specific sequences derived from 3049 and lpD genomes. The portion of the VP1 polypeptide carrying the altered charge could be mapped to the body of the molecule 3' to the HindIII site at 45.0 map units (3,918 base pairs). This clearly segregates the VP1 charge phenotype from the cyc phenotype of 3049 in which capsid proteins are overproduced and accumulate in the cytoplasm of infected cells.

Ampholyte Mixtures↗
PubMed1994

Differentiation by preparative continuous free flow-isoelectric focusing of cyclosporin A inhibitable peptidyl-prolyl cis/trans isomerase of human erythrocytes.

Preparative continuous free flow-isoelectric focusing has been used to separate at least three different components of intrinsic peptidyl-prolyl cis/trans isomerase (PPIase) activity from erythrocytes lysate. By adding chemical spacer molecules like glycine and Bicine to commercial carrier ampholyte mixtures the resulting pH profile was predictably influenced. With an applied field strength of 125-170 V/cm a residence time of less than 15 min was sufficient for the separation of PPIases with isoelectric points of 5.4, 5.7 and 5.9 from the bulky hemoglobin. The recovery of the overall PPIase activities was about 100%. The purification factor has been determined as 20- to 100-fold. For each isoform of the enzyme the peptidyl-prolyl cis/trans isomerase activity of the separated proteins was inhibited by cyclosporin A but was resistant toward FK 506.

Amino Acid Isomerases↗
PubMedDate not supplied

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.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1997

[The effect of keeping condition and genotype on the electrophoretic properties of pork proteins].

Besides differentiation of animal species the electrophoretic methods are efficient also in determinations of the factors effecting the protein patterns. The goal of this study is to compare the effect of the genotype and the keeping conditions on the electrophoretic behaviour of the pork meat proteins. Isoelectric focusing was found to be more efficient than SDS-PAGE concerning studies of the effect of these factors. An optimal separation has been achieved with an ampholyte-mixture at pH ranges 2-11, 5-7 and 7-9. Peroxidase activity of the separated myoglobines of the meat samples was stained for detecting the differences in protein zones according to the keeping conditions. The myoglobin zones stained with o-dianisidin were evaluated by densitometry. The protein zones of the organic meat samples showed higher intensity than those of the control-samples.

Animals↗
PubMed1985

Isotachophoresis of allergenic extracts.

One aspect of the isotachophoretic determination of protein patterns in biological samples of interest is the characterization of allergens. This group of (glyco) proteins, causing allergic reactions, is used both for diagnosis and in the treatment of allergy. The aim of this investigation was to obtain a maximum amount of information, within one run, on the (glyco)protein composition of a number of allergenic extracts (e.g., from pollen or house dust mites). Commercially available extracts were dialysed prior to analysis to remove disturbing buffer constituents. A high-pH system was chosen in order to obtain a maximum amount of information from the samples (1-2 microliter). The leading electrolyte was 0.01 M C1-, buffered with Tris (pH 8.2), containing 0.2% w/v hydroxyethylcellulose, and the terminating electrolyte was beta-alanine, buffered to pH 10 with Ba(OH)2. The total analysis time was 15-20 min using a PTFE capillary (0.2 mm I.D.). The pre-separation current was 30 microA and the current during detection was 15 microA. UV absorption was measured at 280 nm. For optimal discrimination of the compounds of interest, an ampholyte mixture was used for spacing. The analytical procedure yielded highly reproducible UV patterns. Significant differences between various allergenic extracts were observed. It was concluded that isotachophoresis is a powerful method for the physico-chemical characterization of individual allergenic extracts, e.g., with respect to manufacturing and quality control.

Allergens↗
PubMed1980

Visualization of carrier ampholyte patterns in granular gel slabs. 1. Applications to analytical electrofocusing.

1. The isoelectric banding patterns of three commercial carrier ampholytes (pH range 2-11) were visualized in both Sephadex gel slabs and on paper prints, by means of ultraviolet light. 2. The fluorescence pattern of Servalyt is described in detail. This pattern, which has sharply delineated bands, is characteristic, and is reproducible from run to run with different batches of Servalyt. A total of 56 fluorescent bands were identified on gel slabs, and 62 on prints. 3. Methods are described for identifying individual fluorescent bands on prints. A classification scheme for organizing the banding pattern of Servalyt and naming individual bands is presented. 4. On gel slabs and on prints, the fluorescent pattern of a carrier ampholyte may be used analytically as a map for the location and identification of focused protein bands. The utility of the fluorescent pattern for characterising commercial ampholyte mixtures is discussed. 5. Individual fluorescent bands on the gel slab have characteristic pH values, which are constant from run to run and remain constant in the presence of focused sample proteins. On prints, these bands serve as permanent internal markers for assigning isoelectric points to protein bands.

Buffers↗
PubMed1997

Metalloprotein analysis by capillary isoelectric focusing.

Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3-10 and 5-8. Several different proteins and one peptide with known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3-10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe3+/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3-10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (diferric forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5-8). Rabbit liver MT subjected to cIEF with a pH gradient of 3-10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3-10, heterogeneous peaks with higher pI values (4.12-4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using a pH gradient of 5-8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.

Animals↗
PubMed2003

Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization.

In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA(1c), in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 microm I.D., 27 cm, 0.20 microm coating thickness), using a narrow pH ampholyte mixture (4% pH 6-8:pH 3-10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l(-1) phosphoric acid solution was used as anolyte and a 0.040-mol l(-1) NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA(1c), from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l(-1) NaOH solution containing 0.080 mol l(-1) NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA(1c) determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r(2)=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA(1c) concentration higher than the reference method. The separation of the hemoglobins HbA, HbA(2), HbF and HbA(1c) and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA(2) and HbA/HbA(1c).

Calibration↗
PubMedDate not supplied

A new isoelectric focusing gel for two-dimensional electrophoresis constructed in microporous hollow fiber membranes.

We describe the preparation of IEF tube gels inside a nonwetting microporous plastic tubing. The gel in the tube need not be extruded after the first dimension separation. Instead, the porous structure of the tubes is made wettable, and the proteins are electrophoresed "through-the-wall" into the second dimension PAGE gel. Commercial ampholytes and reagents are suitable for the procedure. A useful p/ range of 4.5-9.5 can be obtained when p/ 3-10 ampholyte mixtures are used. Because of the high surface area of the porous material, precautions must be exercised to reduce oxygen inhibition during polymerization and dehydration of the gel during storage and use. A sheath device is described that satisfies these requirements. The plastic tubes can be disposed of by incineration and pose no biohazard.

Animals↗
PubMed2003

Dynamic capillary coatings with zwitterionic surfactants for capillary isoelectric focusing.

The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C12N3SO3), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C16N3SO3) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein-ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C16N3SO3 and CAS U coatings were shown effective in CEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C16N3SO3 was effective in suppressing EOF at acidic and neutral pH conditions. C12N3SO3 however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R2 approximately equal to 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.

Journal Article↗
PubMed1983

Isoelectric focusing in agarose: classification of genetic variants of alpha 1-antitrypsin.

Polyacrylamide has been the matrix of choice for isoelectric focusing owing to the virtual absence of electroendosmosis in this medium. Certain inherent limitations associated with polyacrylamide have prompted some investigators to use low-electroendosmosis agarose for isoelectric focusing, but with limited success thus far. We have developed a method for isoelectric focusing in agarose for the classification of alpha 1-antitrypsin variants. Sera are applied directly to agarose gels containing a pH 4-5 ampholyte mixture, focused for less than 1 h, and directly immunofixed. Resolution of major bands is equivalent to polyacrylamide, and Pi M subtypes can be distinguished without the use of a separator. This application demonstrates the high resolution of isoelectric focusing in agarose, a more practical and convenient matrix than polyacrylamide.

Acrylic Resins↗