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PubMed2004

[Application of denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis in microbial molecular ecology].

In recent years, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are well-established molecular tools in microbial molecular ecology that allows the study of diversity and dynamics of microbial communities. The technique is reliable, reproducible rapid and inexpensive. Here, the principle, the limitations and potentials application of DGGE/TGGE techniques are introduced in this paper.

Bacteria↗
PubMed1982

Two-dimensional gel electrophoresis of human brain proteins. V. Non-equilibrium gel electrophoresis, with detection of a myelin basic protein mutation--MBL-Duarte.

To examine the basic human brain proteins, we subjected 9 mmol/L urea extracts to non-equilibrium gel electrophoresis. The pattern observed differs distinctly from that with equilibrium gel electrophoresis. With this technique, the myelin proteins (myelin basic protein, proteolipids, and basic Wolfgram proteins) and many other unindentified major basic proteins can be demonstrated. The myelin basic proteins occur as two major polypeptides of different charge and slightly different molecular mass, indicating the action of at least two genes. The proteolipid proteins occur as a long series of charge isomers, suggesting multiple genes or extensive post-transcriptional modification. In one patient with schizophrenia, a charge-change mutation of the larger myelin basic protein (MBL) was observed and is termed "MBL-Duarte."

Brain Chemistry↗
PubMed1978

Electrophoresis and immuno electrophoresis in leprosy.

50 cases of leprosy belonging to various subgroups i.e. 10 tuberculoid, 25 lepromatous leprosy, 10 lepra reaction, 5 dimorphic leprosy, and 25 cases or normal individuals were subjected to agar gel electrophoresis. The slides were scanned by densitometry. It showed profound departure from normal in various fractions of electrophoretic patterns. All of them showed rise of gamma globulin. Albumin was markedly decreased in lepromotous leprosy and lepra reaction. In dimorphic leprosy Alfa-1 was decreased. Qualitative immuno electrophoresis was done by using antihuman serum raised in the laboratory, by immunising rabbits. It revealed changes in IgM and IgG arcs. Results are discussed and tried to explain on immunological derangement.

Alpha-Globulins↗
PubMedDate not supplied

Capillary electrophoresis for regulatory analysis: is it ready? Problems encountered during an interlaboratory study utilizing capillary electrophoresis.

Recently, the Northeast Regional Laboratory of the Food and Drug Administration conducted a collaborative study on the method to determine histamine in tuna by capillary electrophoresis. This study, under the guidance of the AOAC International, was the first of its kind involving the use of CE and was undertaken to establish official monograph status. The results of the collaborative study, however, were highly variable. Percent recovery data obtained by 11 collaborators ranged from 54 to 281%, with an average of 111%. In this paper, problems with reproducibility, and factors such as low sensitivity, noisy baselines, and variable migration times encountered by collaborators using various commercial capillary electrophoresis units are discussed.

Animals↗
PubMed1998

Classification of Clostridium butyricum based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis.

Eleven strains of Clostridium butyricum collected from different sources were analysed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pulsed-field gel electrophoresis (PFGE). The strains could be classified into four groups based on their banding profiles of the proteins extracted from the cells on SDS-PAGE. Group I consisted of seven strains, and these strains were further divided into five subgroups by PFGE. The strains belonging to groups II, III, and IV on SDS-PAGE were also classified into the same II to IV groups by PFGE. These data indicate that grouping of the strains of C. butyricum can be performed by employing both SDS-PAGE and PFGE.

Journal Article↗
PubMed2006

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode.

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.

Journal Article↗
PubMed2000

Urine protein electrophoresis and immunofixation electrophoresis supplement one another in characterizing proteinuria.

Urine protein electrophoresis (UPE) is often considered to have limited usefulness in evaluating proteinuria that is not associated with gammopathies. Unusual protein bands that are detected by UPE are commonly characterized by immunofixation electrophoresis (IFE). In this paper, electrophoretic gel patterns are shown to illustrate the greater sensitivity of IFE, compared to UPE. However, UPE remains useful for three applications: (1) UPE provides distinctive patterns that can indicate the source of proteinuria and is useful in assessing renal diseases that are independent of gammopathy; (2) combined use of UPE and IFE can avoid misinterpretations and repeated analyses of urine proteins, and (3) UPE can be used in conjunction with IFE to improve the quantitation of Bence-Jones proteinuria (BJP).

Antigen-Antibody Reactions↗
PubMed1995

Steady-state models in electrophoresis: from isotachoporesis to capillary zone electrophoresis.

Although all electrophoretic techniques are closely allied and controlled by the same rules, we often distinguish between steady-state and dynamic models in the modeling of electrophoretic processes, whereby steady-state models are applied for isotachophoresis (ITP) and dynamic models are applied for other electrophoretic processes, wherein a steady-state is not reached. This paper shows how, starting from a mathematical model for the steady-state in ITP, mathematical models can be derived for several modifications of ITP and that even nonsteady-state processes can be estimated by a repeated application of a steady-state model. In this way all parameters in sample zones in capillary zone electrophoresis (CZE), through which temporal electropherograms can be simulated, can be calculated. Realistic simulations can be obtained for zone electrophoretic processes, for which the electrodispersive character is the dominating peak broadening mechanism, and simulated electropherograms resemble measured electropherograms concerning migration times, peak shapes, fronting or tailing character and the question of peaks and dips.

Computer Simulation↗
PubMed1998

Chemiluminescence detection in integrated post-separation reactors for microchip-based capillary electrophoresis and affinity electrophoresis.

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.

Animals↗
PubMed1994

On the "door-corridor" model of gel electrophoresis. II. Developments related to new gels, capillary gel electrophoresis and gel chromatography.

An excellent separation of small DNA fragments is possible also at a low total polymer concentration. This is illustrated with novel gels containing 1% of the cross-linked agarose polymers. In gel-filled capillaries, preelectrophoresis in the direction opposite to that of separation may create gel discontinuities capable not only of separating, but also of concentrating the migrating molecules. Such capillaries are able to provide an efficiency in the range of 100 x 10(6) theoretical plates per meter. It is proposed that electrophoretic separations in gel-filled capillaries have to be performed at high field strengths in order to achieve the optimal ratio between gel resistance and electrokinetic forces. When considered in terms of the forces exerted on the gel polymers by the migrating macromolecules, gel filtration and gel electrophoresis appear as two profoundly different techniques.

Chromatography, Gel↗
PubMedDate not supplied

Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database.

The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web.

Acrylic Resins↗
PubMed2001

Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry.

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.

Antineoplastic Agents↗
PubMed2001

Capillaries modified by noncovalent anionic polymer adsorption for capillary zone electrophoresis, micellar electrokinetic capillary chromatography and capillary electrophoresis mass spectrometry.

A simple coating procedure for generation of a high and pH-independent electroosmotic flow in capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) is described. The bilayer coating was formed by noncovalent adsorption of the ionic polymers Polybrene and poly(vinylsulfonate) (PVS). A stable dynamic coating was formed when PVS was added to the background electrolyte. Thus, when the PVS concentration in the background electrolyte was optimized for CZE (0.01%), the EOF differed less than 0.3% after 54 runs. The electroosmotic mobility in the coated capillaries was (4.9+/-0.1) x 10(-4) cm2V(-1)s(-1) in a pH-range of 2-10 (ionic strength = 30 mM). When alkaline compounds were used as test substances intracapillary and intercapillary migration time variations (n = 6) were less than 1% relative standard deviation (RSD) and 2% RSD, respectively in the entire pH range. The coating was fairly stable in the presence of sodium dodecyl sulfate, and this made it possible to perform fast MEKC separations at low pH. When neutral compounds were used as test substances, the intracapillary migration time variations (n = 6) were less than 2% RSD in a pH range of 2-9. In addition to fast CZE and MEKC separations at low pH, analysis of the alkaline compounds by CE-MS was also possible.

Adsorption↗
PubMed1989

Toward a steady-state pore limit electrophoresis dimension for native proteins in two-dimensional polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis in linear pore gradients (4.8 to 48% T, 5% CBis) provides for migration arrest, in a practical sense, after about 5000 Vh for proteins of 290 and 450 kDa, but not for smaller proteins over 20,000 Vh. The arrest is not due to inadequate field strength nor is it caused by water redistribution within pore gradient gels. The possibility is being discussed that exponential pore gradients, and a higher or a lower degree of crosslinking suggested by the literature may be remedies for the present failure to arrest the migration of smaller proteins.

Acrylic Resins↗
PubMed1990

Zone broadening in electrophoresis with special reference to high-performance electrophoresis in capillaries: an interplay between theory and practice.

Approximate equations have been derived for the total (final) zone width (plate height, plate number and resolution) as a function of the width of the starting zone and of the zone broadening caused by diffusion. Joule heat, adsorption and the difference in conductivity (delta kappa) between a solute zone and the surrounding buffer. Two cases are treated: (A) the conductivity differences eliminate entirely or (B) partially, the diffusional broadening at one boundary of a zone. When adsorption is negligible one can derive from these equations the field strength - and for case A also the electrical conductivity - that gives the minimum zone broadening (plate height). Interestingly, at this minimum, contributions to the zone broadening from diffusion. Joule heat and conductivity differences have the ratio 4:1:1 in case A. In case B the ratio between the broadening caused by diffusion (including that caused by conductivity and pH differences) and broadening due to Joule heat is 4:1. The total zone width, plate height and optimal field strength calculated from the derived equations agree satisfactorily with experimental values. A simple method to estimate the variance of the zone broadening caused by the Joule heat led to a formula similar to that calculated mathematically. An appropriate width of the starting zone can be calculated rapidly by means of a simple formula. Following a run the true width can be estimated graphically from measurements of plate heights or zone widths at low field strengths. For high resolution the width of the starting zone usually should not exceed 0.5 mm. A new principle for the design of multi-buffer systems which generate sufficiently narrow starting zones has been developed for carrier-free zone electrophoresis. This zone sharpening method permits application of wide zones of concentrations below the detection limit of the monitor. The diffusion coefficient (D) and the universal parameter D/mu (mu = mobility) appear in many of the equations derived and are often the only variables which are not easily accessible. Simple methods have therefore been developed by which they can be determined with sufficient accuracy. Fortunately, they are raised to the power of 1/5 in many formulas and therefore only a rough estimation is required. True plate numbers (calculated in the absence of electroendosmosis) often differ considerably from apparent plate numbers (calculated in the presence of electroendosmosis). A mathematical relationship between the true and apparent plate numbers has been derived.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophoresis↗
PubMed1996

Federated two-dimensional electrophoresis database: a simple means of publishing two-dimensional electrophoresis data.

While a two-dimensional electrophoresis (2-DE) database is a relatively old concept, in recent years it generated renewed interest within the 2-DE community due to two main factors: (i) The high reproducibility of the current 2-DE method allows 2-DE images to be exchanged and compared between laboratories. (ii) The recent development of faster and more powerful techniques for protein identification such as microsequencing, matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and amino acid composition makes the production of reference protein maps and 2-DE databases cost- and time-effective. Additionally, the Internet network's current increase in popularity, combined with the rapid growth of Internet-connected laboratories, provides a straightforward means of publishing and sharing 2-DE data. While a small number of laboratories have already successfully published their data over the net, the increasing number of 2-DE database servers that are currently being set up will sooner or later require some kind of standardization. Unfortunately, standardization can be a long and cumbersome process inevitably leading to undesirable compromises. A federated database offers a simple and efficient way to publish and share 2-DE data without the need for standardization. Taking advantage of Internet protocols such as World Wide Web, they allow each laboratory to maintain their own database and to interconnect it with other similar databases through the use of active cross-references. This paper first presents guidelines for building a federated 2-DE database that may easily be followed by most laboratories. It then briefly reviews the state-of-the-art in networked 2-DE databases, and finally describes the SWISS-2DPAGE database which fully implements the concept of a federated 2-DE database.

Computer Communication Networks↗
PubMed1997

System peaks in capillary zone electrophoresis. 3. Practical rules for predicting the existence of system peaks in capillary zone electrophoresis of anions using indirect spectrophotometric detection.

A theoretical and experimental study of the existence and evolution of system peaks in capillary zone electrophoresis (CZE) with indirect spectrophotometric detection is presented with respect to the effect of the number of coions present in the background electrolyte (BGE). It is shown that in BGEs having only one coion (i.e., the UV-absorbing probe anion), the sample produces only negative peaks due to each analyte anion and no system peaks, with the number of sample peaks corresponding to the number of analytes present in the sample injected. In BGEs containing two coions, a sample with one analyte anion produces one negative indirect detection peak and one system peak. The transition between BGEs having one coion and those with two coions has also been studied and it has been shown that an addition of ca. 5% of the second coion to a single coion BGE causes the resulting BGE to behave macroscopically as a regular two-coion BGE. A descriptive model is proposed, based on transient isotachophoresis (transient stacking) of the sample species and of the coion from the BGE which has the closest mobility to the sample ion. This model explains qualitatively the formation and evolution of the sample peak (containing the sample species and being detected by indirect detection due to displacement of the UV-absorbing probe in its zone) and the system peak (containing no sample species and being a vacancy in the continuum of coions of the BGE). It is shown that the system peak may be positive or negative as it corresponds to the situation where the vacancy of one component of the BGE results in an enhanced concentration of the other component. It has been demonstrated that the system peak is created by a vacancy of that component of the BGE which has the greatest difference in mobility relative to that of the sample species. On indirect detection in BGEs containing two coions the sample displaces predominantly the BGE coion which has a mobility closest to that of the analyte anion. In systems with BGEs containing two coions, a sample having n analytes produces n sample peaks and one system peak, the sign and magnitude of which are dependent on the sum of the UV absorbances of the analytes involved. The effect of bicarbonate from atmospheric CO2 has also been studied and it has been shown that weakly alkaline BGEs with a single anionic UV-absorbing coion, such as those currently used for anionic analyses with indirect detection, may suffer from the presence of system peaks due to bicarbonate.

Anions↗