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PubMed1995

Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems.

After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.

Animals↗

PubMed1995

A new mode of size-dependent separation of proteins by capillary electrophoresis in presence of sodium dodecyl sulfate and concentrated oligomeric dextran.

Capillary electrophoresis in presence of sodium dodecyl sulfate (SDS) and 10% w/v of oligomeric dextran with molecular weight of just above 1000 was found to be an efficient medium for size-dependent separation of proteins (Karim, M. R., Janson, J.-C., Takagi, T., Electrophoresis 1994, 15, 1531-1534). The separations were found to be highly reproducible, both with respect to the position and relative amounts of the peaks. No evidence for a network structure in the medium could be found on examination of the effect of dextran concentration and viscosity measurements. The separation mechanism appears to be different from sieving by a polymer network structure. In free solution, SDS-protein complexes with molecular weights > 10,000 migrate with almost the same mobilities, irrespective of their molecular weight (this behavior is called free draining). Such SDS-complexes of proteins or peptides with molecular weights < 10,000, however, tend to increase their negatively signed mobilities with decreasing molecular weight (Karim, M. R., Shinagawa, S., Takagi, T., Electrophoresis 1994, 15, 1141-1146). Our working hypothesis is that in presence of the oligomeric dextran, the range of deviation from the free-draining electrophoretic behavior extends to higher molecular weights, resulting in a novel separation made.

Circular Dichroism↗

PubMed1995

Capillary electrophoresis with polymeric separation media: considerations for theory.

The theory of capillary electrophoresis with polymeric separation media is reviewed. Some of the major theories of gel electrophoresis are examined in terms of the experimental observations that they support. The considerations and difficulties in applying these theories to capillary electrophoresis with polymeric separation media are investigated, particularly the use of high electric field strengths and low concentration polymer solutions. Recent advances in this area, including some empirically-derived relationships and models currently found in the literature, and future directions for theory development are discussed.

Electrophoresis, Capillary↗

PubMed1995

Band width measurement in automated gel electrophoresis apparatus: DNA dispersion in a discontinuous system and in a single buffer.

Recent commercial introduction of automated gel electrophoresis apparatus allows for band width measurements during electrophoresis and therefore promises to open up the exploitation of band width and shape for the physical characterization of charged macromolecules in the same manner in which to date quantitative gel electrophoresis had exploited electrophoretic mobility at multiple gel concentrations. The measurements demonstrate decreased band width and therefore increased resolving power for a discontinuous buffer system compared to Tris-borate EDTA buffer. The dispersion coefficients (D' = (sigma 2-sigma 2o)/t) of homogeneous DNA components appear to decrease with gel concentration when either the field strength or the DNA length is small, and increase with gel concentration when these are large. This contrasting response of D' to increasing gel concentration is presumably due to DNA stretching, which increases in proportion to DNA length and field strength, and to the progressive orientation of agarose with increasing field strength.

Autoanalysis↗

PubMed1995

Application of capillary electrophoresis to amino acid sequencing of peptide.

Separations of twenty phenylthiohydantoin (PTH) amino acids, and amino acid derivatives, resulting from the Edman degradation of peptides and proteins, were optimized for peptide sequencing by capillary electrophoresis. Manual sequencing of angiotensin II was performed by Edman degradation and capillary electrophoresis of the PTH amino acid obtained after each cycle. The results were compared with those of an automated conventional protein sequencer. Interfacing capillary electrophoresis with Edman degradation provides an additional option for protein sequencing.

Amino Acid Sequence↗

PubMed1996

Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity.

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.

DNA↗

PubMed1996

High resolution density gradient electrophoresis of cellular organelles.

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 x 2.2 cm) containing a 0-5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O-ring. The top circular platinum anode allows for upward electrophoresis (80-100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle-enclosed enzymes.

Cellulose↗

PubMed1996

Sodium chloride in preparative free-flow cell electrophoresis: recent developments.

Three buffer systems for free-flow electrophoresis have been designed, which proved useful for performing cell electrophoresis in the presence of 50 mM NaCl. Each system consists of one central cell suspension buffer with 50 mM NaCl, two margin buffers, and two electrode buffers. With the aid of a bromophenol blue/maxilon blue accumulation test the various buffers were adjusted to ensure a laminar flow and remain unchanged on their way through an electrophoresis chamber.

Buffers↗

PubMed1996

Identification of different galectins by immunoblotting after two-dimensional polyacrylamide get electrophoresis with immobilized pH gradients.

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

Brain Chemistry↗

PubMed1996

An exactly solvable Ogston model of gel electrophoresis: I. The role of the symmetry and randomness of the gel structure.

The Ogston-Morris-Rodbard-Chrambach model (OMRCM) of gel electrophoresis assumes that the mobility (mu) of charged particles is directly proportional to the fractional volume (f) of the gel that is available to them. Many authors have studied the fractional volume f in detail for various particle shapes, but the original assumption, that mu sf, has not been scrutinized seriously. In fact, this geometrical model of electrophoresis does not take into account the connectivity of the gel pores or the precise gel architecture. Recently (G. W. Slater and H. L. Guo, Electrophoresis 1995, 16, 11-15) we developed a Monte Carlo computer simulation algorithm to study the electrophoretic motion of simple particles in gels in the presence of fields of arbitrary strength. Our preliminary results indicated that the mobility and the fractional volume were not generally proportional to one another. In this article, we show how to calculate, in the limit where the field intensity is vanishingly small, the exact electrophoretic mobility of particles in any type of gel in two or more dimensions. Our results, presented here for some simple two-dimensional systems, indicate that a particle can have different electrophoretic mobilities in gels in which it has access to the same fractional available volume f. The curvature of the Ferguson plot is shown to be related to the symmetry and the degree of randomness that characterize the gel. We also demonstrate that the OMRCM is, in fact, a mean field approximation that corresponds to a uniform, annealed gel. We thus conclude that the relation between the electrophoretic mobility and the gel concentration (C) is a delicate function of the gel architecture, and that one needs more than the fractional volume f to fully characterize the transport properties of migrating particles in separation media. Exact relationships between the mobility mu and the gel concentration C are given for our model gels.

Animals↗

PubMed1996

Gel and free solution electrophoresis of variably charged polymers.

To assess the role of charge density on polyelectrolyte mobility, both gel and free solution electrophoresis experiments are performed on poly(acrylic acid) and acrylic acid/acrylamide copolymers. Control of charge density for poly-(acrylic acid) is achieved through solution pH, while control for acrylic acid/ acrylamide copolymers is obtained through chain composition. In either approach, the effective fraction of charged repeat units can be varied from 0 to 100% without a major interruption of solvent quality. Polyelectrolyte mobility in the presence of a monovalent counterion is observed to rise linearly with charge density when this density is low. A transition to charge density independence then occurs over a surprisingly narrow window of charge density. For vinyl polymers of the sort examined here, the transition occurs when 35-40% of the repeat units are charged. These observations are qualitatively consistent with the free solution electrophoresis model proposed by Manning and several previous data sets. An unexpected overlap of normalized gel and free solution data reveals that the charge density exerts a comparable influence in either environment. Results from the present study help define the experimental conditions in which electrophoresis can provide polymer separation by charge density and those in which the method can provide polymer separation by molecular weight.

Acrylates↗

PubMed1996

An exactly solvable Ogston model of gel electrophoresis. II. Sieving through periodic gels.

Recently, we developed a lattice model to study the dynamics of particles being electrophoresed in gels (G. W. Slater, H. L. Guo, Electrophoresis 1995, 16, 11-15). In Part I of this series (G. W. Slater, H. L. Guo, Electrophoresis 1996, 17,977-988), we showed how to calculate the exact electrophoretic mobility of one-site particles in the limit where the electric field intensity E is vanishingly small. Since we can solve the model for arbitrary gel structures in two or more dimensions, we compared our results with those of the Ogston-Morris-Rodbard-Chrambach model (OMRCM) of gel electrophoresis, which assumes that the mobility (mu) of charged particles is directly proportional to the fractional gel volume (f) that is available to them. Our results and theoretical analysis indicated that the OMRCM is a mean-field approximation that can be useful as a rough guide; however, it generally misses the subtle sieving effects related to the correlations between the position of the obstacles in a given gel structure. In this paper (Part II) we study, for two-dimensional periodic gels, the exact relationships between the zero-field mobility mu and the gel concentration C for larger particle sizes. The fact that mu is a strong function of the particle size suggests that we can separate large particles using two-dimensional periodic gels (similar to those fabricated by W.D. Volkmuth and R.H. Austin, Nature 1992, 358, 600-602). We analyze our data using Ferguson-like plots and we show that one can indeed use a generalized retardation coefficient, K, to estimate the effective pore size aK and effective fiber size rK for these model gels. We conclude that the retardation coefficient is a useful concept to characterize a sieving structure even though it does not permit the inference of the exact gel structure.

Electrochemistry↗

PubMed1996

Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light-harvesting complex (LHC II).

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light-harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0-10 with running electrolyte solutions containing either anionic, zwitter-ionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n-octyl beta-D-glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resolution of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.

Chloroplasts↗

PubMed1997

Factors that affect the stability of protein-DNA complexes during gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free-solution lifetimes. Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis. We have used the well-characterized interaction of the E. coli cyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP-DNA complexes. In addition, we found that a cross-linked gel matrix is not required for the stabilization of CAP-DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers. While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein-DNA complexes, they are all features expected of excluded volume mechanisms.

Acrylic Resins↗

PubMedDate not supplied

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis.

Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post-translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two-dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two-dimensional electrophoresis of alkaline phosphatase-treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post-separation analysis, micropreparative two-dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4-7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)-separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2-4.9, 4.5-5.4, 5.0-6.0 and 5.6-6.6.

Animals↗

PubMed1997

Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis.

Rat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen-specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197-208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich-technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well-preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolation of peroxisomal subpopulations that are difficult to obtain by means of density gradient centrifugation.

Animals↗

PubMed1997

Capillary electrophoresis of pesticides: V. Analysis of pyrethroid insecticides via their hydrolysis products labeled with a fluorescing and UV absorbing tag for laser-induced fluorescence and UV detection.

Some representative standard pyrethroid insecticides, namely permethrin, phenotrin, cypermethrin, sanmarton and fenpropathrin, were subjected to base hydrolysis with the aim of facilitating the indirect determination of these neutral species of low water solubilities by aqueous capillary electrophoresis. This first involved the base fragmentation of the pyrethroids in alcohol buffer (pH 12.0), and then the selective tagging of the carboxylated hydrolytic products with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) via a condensation reaction in the presence of organic soluble carbodiimide. The tagging of the hydrolytic products with ANDSA imparted each of the derivatives with two strong sulfonic acid groups whose permanent charges were necessary for achieving aqueous capillary electrophoresis. In addition, the labeling with ANDSA allowed the detection of the derivatives at low levels by capillary electrophoresis laser-induced fluorescence. The geometric and optical isomers of the ANDSA derivatives of the pyrethroid hydrolytic products were best separated when using electrolyte systems composed of sodium phosphate buffer, pH 6.5, containing n-octylglucoside chiral surfactant in the presence of small amounts of acetonitrile (e.g., 10% v/v).

Electrophoresis, Capillary↗

PubMed1997

Clinical application of capillary electrophoresis to unconcentrated human urine proteins.

Urine protein electrophoresis can be performed by capillary electrophoresis using spun unconcentrated urine diluted with running buffer. The separation which utilises the excellent sensitivity of capillary electrophoresis gives electropherograms similar to those obtained by diluting concentrated urine with buffer. A 72 cm x 50 microns internal diameter (ID) fused silica capillary was used for the analysis which took less than 15 min. Bence Jones protein can be detected from unconcentrated urine over urine protein concentrations ranging from 9.7 g/L to 0.04 g/L. Other clinical patterns, such as glomerular proteinuria, the presence of intact immunoglobulin and Tamm Horsfall protein can also be detected. The benefit of using unconcentrated urine is the time and cost saved by not concentrating the urine.

Adult↗