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At least 109 records · Page 6Linked to original sources

PubMed1990

Two-dimensional polyacrylamide gel electrophoresis of human hair matrix proteins using isoelectric focusing electrophoresis as the first dimension.

S-carboxymethylated (SCM) matrix proteins from fifteen normal human scalp hair specimens were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE) with usual isoelectric focusing electrophoresis as the first dimension and then with usual sodium dodecyl sulfate-PAGE as the second dimension. On the electrophoregrams obtained, one low-mobility smear of SCM proteins embedding one SCM protein band was always seen. Many higher-mobility SCM protein spots were also always seen. However, some variations were seen in these SCM protein compositions; in type I, there were eighteen SCM protein components and, in type II, seventeen components.

Adult↗

PubMed1990

Column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of pig lens crystallins.

An identical size of porcine and bovine lens crystallins could be demonstrated using gel chromatography, while slight differences were found in their relative concentrations. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both types of lens crystallins were found very similar, with the only exception of a slightly lower molecular weight of the porcine beta-h and beta-i subunits. By two-dimensional electrophoresis, 14 out of 19 spots likewise coincided. This great similarity in crystallin subunits between both animals agrees with their phylogenetic proximity, and it is suggested that porcine and bovine lenses may be used in very similar ways for investigation of posttranslational modifications of crystallins.

Animals↗

PubMed1986

Two-dimensional gel electrophoresis, isoelectric focusing and agarose gel electrophoresis in the diagnosis of multiple sclerosis.

Two dimensional gel electrophoresis (2-DE), isoelectric focusing (IEF) and agarose gel electrophoresis (AGE) were used to examine cerebrospinal fluid (CSF) and sera from 22 patients with confirmed multiple sclerosis, 11 patients with probable multiple sclerosis and 20 control patients with non-inflammatory neurological diseases of the central nervous system (CNS). All of the 22 patients with confirmed multiple sclerosis showed abnormal patterns of oligoclonal IgG in all three methods. In the CSF from patients with probable multiple sclerosis, oligoclonal IgG was detectable in 18 percent with AGE, in 72 percent with IEF and 90 percent with 2-DE. No oligoclonal IgG was observed in subjects with non-inflammatory neurological diseases. Many artefacts in IEF, which lead to misinterpretation, are eliminated in the 2-DE system. Based on our observations and this study in particular, it is evident that some patients have IgG changes which can be detected only by 2-DE. The application of research-oriented 2-DE for routine clinical purposes is still limited by its cost and technical complexity.

Electrophoresis, Agar Gel↗

PubMed2001

From paper electrophoresis to computer-supported interpretation of capillary electrophoresis--clinical plasma protein analysis in Malmö, Sweden.

Protein analyses have been used in Malmö as a routine clinical diagnostic tool since 1953. Most serum samples are submitted for "protein profiles" including capillary zone electrophoresis and rate immune nephelometric quantification of nine proteins (five in urines), although analysis of single proteins may be requested. Standardization between laboratories in our region has been greatly improved by automation, CRM 470 calibration and external quality assurance. We are further extending standardization by developing computer supported interpretations using a program with improved user interface and graphical representation of electrophoretic curves superimposed upon a shaded reference interval. Programming is underway to provide complete automatic interpretation of these curves. Together, capillary electrophoresis (with access to mathematical analysis) and immunochemical quantifications allow a highly automated process accessible to further digital analysis and automated interpretation. Rapid, cost-effective and standardized analysis of serum protein profiles should improve the diagnostic evaluation of many categories of patients.

Blood Proteins↗

PubMedDate not supplied

A comparative study of fish species identification by gel isoelectrofocusing two-dimensional gel electrophoresis, and capillary zone electrophoresis.

A comparative study of fish species identification was accomplished using three different electrophoretic techniques. Sarcoplasmic proteins were extracted from three related fish species and subjected to gel isoelectrofocusing (IEF), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and capillary zone electrophoresis (CZE). The fish species--Genypterus chilensis, Genypterus blacodes, and Genypterus maculatus--were from the Ophidiidae family. The three electrophoretic techniques provided suitable fish species identification. Nevertheless, CZE demonstrated several advantages over the other two conventional techniques. Some of the benefits include the use of small amounts of reagents; short separation times, permitting fast comparative analysis; data reproducibility; and ease with which the technique is performed.

Animals↗

PubMed1994

Analysis of snake venoms by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis.

The protein composition of snake venom has been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE) with comparison of our simplified Tris/glycine/SDS (TGS) method with a commercial Tris/Tricine/SDS (TTS) gel system. SDS-PAGE suggests differences between species and similarities between related species but 2-DE indicates that the protein composition of the venoms is highly complex. The TTS system improves resolution upon 2-DE but our simplified TGS method is optimal for silver staining which, contrary to previous reports, enhances detection 100-fold.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2000

Blood compatibility of particulate carriers investigated by cell electrophoresis and particle electrophoresis: a preliminary report.

Polyethylene glycol (PEG) and dextran were covalently coupled, or only adsorbed, to the surface of three kinds of inorganic particles in order to shield their surface and reduce their nonspecific binding to red blood cells. Surface modification as well as interaction of particles with red blood cells was followed up by particle electrophoresis. This allowed a quick evaluation of the efficiency of polymer coupling. Moreover, the nonspecific binding of particles to red blood cells was easily investigated with cell electrophoresis, showing the inhibitory effect of immobilized PEG-5000 or dextran. The electrophoretic mobility analysis presented here may be used for screening blood compatibility of particulate drug carriers and could be helpful in formulating long-living circulating particles.

Animals↗

PubMed2002

Up-scaling capillary zone electrophoresis separations of polydisperse anionic polyelectrolytes with preparative free-flow electrophoresis exemplified with a soil fulvic acid.

A scale-up of analytical capillary zone electrophoresis (CZE) to preparative free-flow electrophoresis (FFE) is described. FFE allows fractionations based on charge densities in larger amounts than in CZE, enabling further off-line analysis of the fractions. Model compounds (carboxylic acids and polystyrene sulfonates) showed a similar behavior in FFE as in CZE. Diffusion and electrodynamic distortion effects are more pronounced in FFE than in CZE. A soil fulvic acid was analyzed by CZE and fractionated by FFE. A comparison of the FFE fractions with CZE measurements of the same sample using the effective mobility scale showed good agreement of the two methods.

Benzopyrans↗

PubMed1988

Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem.

PhastSystem, an integrated system for horizontal electrophoresis and isoelectric focusing in small gels, including automated staining and destaining, is described. Buffers for electrophoresis are supplied to the gel from buffer strips made of agarose. The separation bed is cooled by Peltier elements. All conditions of significance to the results, both during separation and development, are controlled by a microprocessor. For separation, nine programs are available, each with 9 steps; for development, there are nine programs with 20 steps each. In this paper, we also report results using PhastSystem for purity checking and characterization of monoclonal antibodies after affinity chromatography and also for determining their digestion rate with papain.

Buffers↗

PubMed1990

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis.

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.

Bacterial Proteins↗

PubMedDate not supplied

High speed electrophoresis simulation for optimization of continuous flow electrophoresis and high performance capillary techniques: Part I. Computer model.

A computer program for high-speed simulation and optimization of electrophoretic processes has been developed for carrier-free systems of all kinds. The calculations are based on the one-dimensional dynamic (transient-state) model. The three-dimensional geometry of the simulation space can be chosen deliberately. With a highly efficient transport algorithm instead of complicated integration schemes for the transport equations, the calculation time can be effectively spent on various important parameters such as ionic strength, temperature, Joule heat, activity coefficients and concentration changes due to membranes. The parameter set of any carrier free electrophoretic method (i.e., continuous-flow electrophoresis, capillary isotachophoresis and high performance capillary zone electrophoresis) can be imported directly into the computer program by means of a graphic user interface. The program performs overnight-simulation of any electrophoretic system containing up to 15 components.

Computer Simulation↗

PubMed1997

Isolation of subcellular-sized particles separated by electrophoresis in dilute polymer solution, using commercial electrophoresis apparatus with intermittent scanning of fluorescence.

Resolution of subcellular-sized particles in electrophoresis employing semi-dilute polymer solutions as "sieving media" improves as the polymer concentration is decreased. Therefore, the previously reported conditions of preparative electrophoresis of microsomes, using concentrated (12%) polyvinylpyrrolidone (PVP) solutions, while solving the problem of non-entrance of large particles into "sieving media", do not provide adequate resolving capacity, as exemplified by failure of the microsome preparation used, to resolve in the manner of gels or dilute solutions. The present report provides the conditions under which the HPGE-1000 apparatus can be preparatively applied when the electrophoretic separation is effectively conducted in a dilute polymer solution. The isolation of three microsome components under those conditions constitutes the first application of "particle sieving", i.e., a separation due preponderantly to size and shape differences, at a preparative scale.

Animals↗

PubMed2003

Capillary electrophoresis--electrospray ionization--mass spectrometry for the characterization of natural organic matter: an evaluation with free flow electrophoresis-off-line flow injection electrospray ionization-mass spectrometry.

The separation of Suwannee River natural organic matter (NOM) with capillary zone electrophoresis hyphenated to electrospray ionization-mass spectrometry (CZE-ESI-MS) is presented. The obtained electropherograms and signal distributions are comparable to the mobility distributions obtained with more classical UV detection. A direct comparison of the results was possible with free-flow electrophoresis (FFE), which allows an upscaling of the CZE method and the analysis of the collected fractions in an off-line modus with flow-injection electrospray ionization-mass spectrometry (FI-ESI-MS). The changes of the m/z distributions with mobility are very similar with both methods and show a decrease of the m/z with increasing electrophoretic mobility in the humic hump at alkaline pH; superimposed on this hump a low-molecular-weight fraction migrates at lower mobility. The analysis of benzene carboxylic acids, glycerrhycic acid as well as oligomers of polystyrene sulfonic acid and polyacrylic acid additionally illustrates possible fragmentation, formation of adducts and multiplicity of the charges of the molecules prior to MS detection. These hardly controllable difficulties add a challenge to the interpretation of the obtained m/z distributions of NOM in terms of charge and mass distributions of molecules present in the NOM mixture.

Acrylic Resins↗

PubMed2004

Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate.

We report the one- and two-dimensional (1-D and 2-D) capillary electrophoresis separation of Deinococcus radiodurans protein homogenate. Proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), which reacts with lysine residues and creates a highly fluorescent product. Detection is by laser-induced fluorescence. 1-D capillary sieving electrophoresis (CSE) produces over 150,000 plates and micellar electrokinetic capillary chromatography (MEKC) produces over 900,000 plates for components in a D. radiodurans protein homogenate. In a 2-D separation, proteins are first separated by CSE. Fractions are repetitively transferred to a second capillary for further separation based on MEKC. The 2-D separation has a approximately 550 spot capacity. Over 150 components are partially resolved from the homogenate. Resolution is limited in the first dimension by diffusion of proteins during the long separation period and in the second dimension by the combination of a long fraction-transfer time and short separation period.

Automation↗

PubMed2005

Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Animals↗

PubMed1992

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Animals↗

PubMed1988

Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis.

A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Schägger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.

Electrophoresis, Polyacrylamide Gel↗

PubMed1975

Micrococcus lysodeikticus ATPase. Purification by preparative gel electrophoresis and subunit structure studied by urea and sodium dodecylsulfate gel electrophoresis.

Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its "shodk wash" release from the membrane. The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures. The pure protein had a specific activity of 7 mumol Pi-min- minus 1-mg- minus 1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin. By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sokium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (alpha and beta) and two minor ones (gamma and delta). The non-identity between the major subunits was demonstrated.

Adenosine Triphosphatases↗