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At least 19 recordsLinked to original sources
PubMed1987

Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.

A characteristic feature of high-performance electrophoresis (HPE), the electrophoretic counterpart of high-performance liquid chromatography (HPLC), is that the separation chamber is a thin-walled, narrow-bore (0.05-0.3 mm) glass or fused-silica capillary tube for rapid dissipation of the Joule heat in order to minimize thermal zone deformation even at high field strengths. This paper is centered around the usefulness of HPE for separation in a carrier-free medium (i.e., in buffer alone) and deals with both zone electrophoresis, isoelectric focusing and displacement electrophoresis. Examples are given of analytical and micropreparative separations of inorganic and organic ions, proteins, viruses and bacteria. The run times are 5-30 min. Discontinuous buffer systems have up to now been used exclusively for the separation of proteins by electrophoresis in polyacrylamide gels ("disc electrophoresis"). However, the Ornstein and Davis discontinuous buffer system has been modified to adapt it to carrier-free zone electrophoresis in order to achieve automatic sharpening of the starting zone. Very high resolution of serum proteins was obtained when they were subjected to free high-performance disc electrophoresis in such a modified buffer system. To show that the HPE apparatus permits electrophoresis also in a gel medium, a polyacrylamide electrophoresis in SDS is presented. This experiment illustrates the difference between electropherograms obtained in free solution and in a molecular-sieving medium. Detection can be performed both on- and off-tube. The latter technique permits the rapid identification of the solutes by photodiode array spectrophotometry and the collection of fractions for further studies. The former detection method is simpler but mainly useful for analytical purposes. Non-UV-absorbing ions can be monitored with the aid of an on-tube UV detector if the run is performed in a UV-absorbing buffer.

Blood Proteins↗
PubMed1990

Scale-up of free-flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step-zone electrophoresis.

Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step-zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8-fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step-zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1-fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale-up to 3 g/h is possible in zone electrophoresis and field step electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1998

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis.

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins-Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins-Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.

Blood Proteins↗
PubMed1997

Conventional capillary electrophoresis in comparison with short-capillary capillary electrophoresis and microfabricated glass chip capillary electrophoresis for the analysis of fluorescein isothiocyanate anti-human immunoglobulin G.

Fast, efficient analysis of fluorescein isothiocyanate anti-human IgG was achieved in a short-capillary and in a microfabricated glass chip. The capillary was 6 cm long from injection end to detector with electric field strength of 0.268 kV/cm. Analyses were performed within 1 min. A glass microchip device was fabricated using standard photolithographic procedures and chemical wet etching. The channels were sealed using a direct bonding technique. For an effective length of 2.8 cm with electric field strength of 0.526 kV/cm, electrophoretic analysis was achieved in less than 16 s. Conventional capillary electrophoresis gave highly reproducible results and good detection limits. Analysis time was 2.5 min for a capillary with 47 cm effective length and electric field strength of 0.383 kV/cm.

Electrophoresis, Capillary↗
PubMed1996

Capillary electrophoresis of serum proteins. Reproducibility, comparison with agarose gel electrophoresis and a review of the literature.

Conditions of serum protein analysis by capillary electrophoresis were optimized and within day, between day and between capillary variations were examined for both migration times and relative peak areas. For the five currently accepted zones, albumin, alpha 1, alpha 2, beta and gamma-globulin, reproducibilities of migration times were in the range of 2.3-3.1% (n = 200 measurements). Although variations in relative peak areas were slightly higher than those obtained by conventional agarose gel electrophoresis, from a resolution perspective, capillary electropherograms provided better detail than the densitometric scans of agarose gel electrophoresis. Precise localization of C3 and transferrin in capillary electrophoresis resulted in more accurate detection of the beta-globulin fraction. When C3 appeared in the gamma-fraction it was not detected as a separate peak in agarose gel electrophoresis, whereas it was in capillary electrophoresis. In artificially prepared mixtures of highly purified albumin and gamma-globulin preparations, best correspondence with theoretical values was found with capillary electrophoresis. Inter-individual variations and reference values were obtained by measuring 140 samples from healthy controls (59 females, 81 males) with both techniques. For capillary electrophoresis the inter-individual variations of the albumin, alpha 1, alpha 2, beta and gamma fractions were respectively 6, 21, 19, 14 and 18% and for agarose gel electrophoresis 5, 20, 17, 18 and 22%. From these results it can be concluded that the more precise localization of the beta- and gamma-globulin fraction results in about 4% lower inter-individual variations in capillary electrophoresis compared to agarose gel electrophoresis. For the other fractions, comparable variations were obtained. Differences between males and females were not significant. For patient samples, a good correlation was found between capillary electrophoresis and agarose gel electrophoresis data for all five protein fractions. We conclude that separation efficiency of capillary electrophoresis is better than that of agarose gel electrophoresis and even weak monoclonal components can easily be distinguished with the capillary electropherogram. Capillary electrophoresis is a qualitatively good, cheap, fast and easy to perform alternative to agarose gel electrophoresis.

Blood Proteins↗
PubMed2000

Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis.

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.

Chlorpheniramine↗
PubMed1997

Characterization of thioredoxins by sodium dodecyl sulfate-slab gel electrophoresis and high performance capillary electrophoresis.

Disulfide containing proteins--thioredoxins from E. coli and pig heart mitochondria--were characterized by sodium dodecyl sulfate (SDS)-electrophoresis and high performance capillary electrophoresis (HPCE). Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS-electrophoresis, and sample storage. All these factors influenced the relative intensities of several protein bands with thioredoxin-like mobility, whereas the sample storage also resulted in the appearance of SDS- and dithiothreitol (DTT)-resistant high molecular mass forms, probably thioredoxin dimers. The multiple forms of the thioredoxin from pig heart mitochondria in SDS-electrophoresis might be dependent on the oxidation state of the protein cysteine residues. A commercial preparation of the thioredoxin from E. coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or without DTT. After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS-electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay. Also, thioredoxin from E. coli, homogeneous on SDS-electrophoresis, was found heterogeneous on HPCE. The peak corresponding to the insulin-dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE.

Animals↗
PubMed1994

Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.

Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis.

DNA↗
PubMed1975

Concanavalin A-reactive protein of rabbit thymocyte plasma membranes: analysis by crossed immune electrophoresis and sodium dodecylsulfate/polyacrylamide gel electrophoresis.

1. Thymocyte plasma membrane extracts, prepared with the non-ionic detergent Triton X-100, show 10 major protein components upon sodium dodecysulfate/polyacrylamide gel electrophoresis and at least 11 immunologic components upon crossed immune electrophoresis. 2. Concanavalin A reactive membrane proteins have been identified using crossed immune electrophoresis with receptor-ligand interaction. 3. These proteins are absorbed from Triton X-100-solubilized membranes onto immobilized concanavalin A. They are eluted in stepwise fashion, using increasing concentrations of alpha-methyl-d-glucoside, between 0.0004 M and 0.1 M. The predominant proteins eluted in each step are components with high electrophoretic mobility in crossed immune electrophoresis and are identical with a glycosylated component in sodium dodecysulfate/polyacrylamide gel electrophoresis with molecular weight of 55 000. 4. This component forms multimers in the presence of Triton X-100 which are not totally dissociated in sodium dodecylsulfate. 5. Neuramidase treatment followed by crossed immune electrophoresis of total plasma membrane isolates, as well as the purified glycoprotein fraction, indicates that the concanavalin A-reactive proteins are sialoglycoproteins. 6. Sodium dodecylsulfate component 5.1 comprises at least two different populations of glycoproteins (6 and 9) in crossed immune electrophoresis, one of which exclusively exhibits heterogenous carbohydrate antigenic sites (component 9). 7. Present data, taken together with previously published experiments, indicate that concanavalin A binding to intact thymocytes induces an increased turnover and release of the receptor protein(s).

Animals↗
PubMed1994

Sponge-like electrophoresis media: mechanically strong materials compatible with organic solvents, polymer solutions and two-dimensional electrophoresis.

A new range of sponge-like media, called "electrophoresis sponges", is presented. They differ from electrophoresis gels primarily in that they are mechanically stronger, providing a permanent structure of directly measurable pore size dimensions. The new media are similar to capillary electrophoresis in terms of pore size range, they are mechanically strong with directly definable walls, and are compatible with polymer solutions. The sponges differ from capillary electrophoresis in that they provide large numbers of channels, with a corresponding high load capacity for simultaneous runs in multiple channels and they are compatible directly with multi-dimensional separations, such as high resolution two-dimensional electrophoresis. Furthermore, they can be molded (or cut) to any shape and retain that shape, they can be handled more easily than gels, they can be reused if necessary, they can be distributed in the same format between labs easily, and they can be stored indefinitely. Chemically, they can be hydrophilic or hydrophobic, with capability ranging from inert to reactive surfaces. Pore sizes can range from the sub-nanometer to 100 micron scale. Results with various hydrophobic sponges are reported for the carrier ampholyte-based isoelectric focusing of proteins. Broad and narrow pH gradients are established in the sponges that are more linear than those achieved with polyacrylamide gels. One- and two-dimensional electrophoresis of proteins has been achieved, for example with high resolution of the charge isomers of the haptoglobin beta chain, using sponge-based isoelectric focusing. Isoelectric focusing is about threefold faster in the tested sponges than in equivalent polyacrylamide gels. This improved speed is probably related to the larger sponge pores.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Gel, Two-Dimensional↗
PubMed1995

Comparison of field inversion gel electrophoresis with contour-clamped homogeneous electric field electrophoresis as a typing method for Enterococcus faecium.

Direct comparisons between contour-clamped homogeneous electric field (CHEF) electrophoresis and field inversion gel electrophoresis (FIGE) to determine the epidemiology of antibiotic-resistant enterococci have not been previously published. Fifty non-beta-lactamase-producing, ampicillin-resistant Enterococcus faecium isolates and 10 vancomycin-resistant E. faecium strains collected from multiple centers were analyzed in a blinded fashion by CHEF electrophoresis and FIGE after digestion with SmaI. Isolates were considered clonally related if there was a difference of three of fewer bands between electrophoretic patterns. Agreement between CHEF electrophoresis and FIGE was seen for 12 of 13 identified groups of ampicillin-resistant E. faecium and 7 of 7 groups of vancomycin-resistant E. faecium. The lone discordance was accounted for by a fourth band difference between two strains recognized near 350 kb by CHEF electrophoresis but not by FIGE, placing them into different clonal groups. Better band separation was noted in the 50- to 200-kb range for FIGE, while CHEF electrophoresis revealed better resolution over 250 kb more reliably, including detection of some bands not seen on FIGE. Molecular epidemiologic investigations of E. faecium by either technique should provide comparable results.

Ampicillin Resistance↗
PubMed1998

Differential diagnosis of gammopathies by capillary electrophoresis and immunosubtraction: analysis of serum samples problematic by agarose gel electrophoresis.

The capabilities of capillary electrophoresis (CE) for serum protein electrophoresis and immunotyping have been demonstrated. CE-based systems specifically designed for serum protein electrophoresis and immunotyping via immunosubtraction (IS) are now available and are being evaluated for efficiency, specificity and sensitivity by several groups. The use of CE for serum protein electrophoresis and immunotyping (IS) in the clinical laboratory compares well with agarose gel electrophoresis (AGE) and immunofixation (IF) for the detection and characterization of monoclonal proteins. In addition to routine use, this technology is useful for a subset of serum samples that are difficult to interpret with conventional technology. In this study, sera abnormalities difficult to detect/interpret by AGE-IF are subdivided into four categories: (i) patients with polyclonal increases in immunoglobulin, (ii) point of application artifacts, (iii) abnormalities in the beta region, and (iv) patients with free light chains. CE is superior to AGE for evaluating samples characterized by the above abnormalities. Sera containing monoclonal proteins within a polyclonal increase are easier to detect by CE as well as being easier to type by IS than by IF. Point-of-application artifacts, periodically observed with AGE, do not exist on CE since the point of detection is remote from the point of application. Enhanced resolution in the beta region allows for increased detection of monoclonal proteins migrating in this region. Some free light chains are undetected by CE as a result of no apparent abnormalities on the CE serum protein profile and, thus, still require IF for detection. CE detects more serum electrophoretic abnormalities than AGE in this clinically important group of patients with Bence Jones proteinemia.

Antibodies, Monoclonal↗
PubMed1996

The use of a new gel matrix for the separation of DNA fragments: a comparison study between slab gel electrophoresis and capillary electrophoresis.

TreviGel-500, a new polysaccharide matrix containing AgaCryl, commercially available as a powder for slab gel electrophoresis, is now being applied to the separation of DNA fragments in capillary electrophoresis. The capillary mode allows the use of one to two orders of magnitude lower mass fractions of matrix and approximately five to six orders of magnitude lower sample quantities than the slab gel electrophoresis counterpart for optimal separation of DNA fragments in the 100 to 2,000 base pair size range. In the capillary mode, this new separation matrix forms a semi-rigid gel that demonstrates enhanced selectivity for DNA fragments in the 1,000 to 7,000 base pair size range relative to alternative size-sieving polymer solutions. In addition, this matrix offers the advantages of lower toxicity than acrylamide. Comparisons are drawn between the use of this matrix in both slab gel electrophoresis and capillary electrophoresis for the separation of DNA fragments with respect to the mass fraction of the matrix in buffer, the buffer composition and sample loading or injection parameters.

DNA↗
PubMed1998

Serum protein electrophoresis and immunofixation by a semiautomated electrophoresis system.

Semiautomated agarose electrophoresis and immunofixation performed with Hydrasys-Hyrys (Sebia) were compared with conventional, manually performed methods, including cellulose acetate electrophoresis, immunoelectrophoresis, and immunofixation. Reference intervals for agarose electrophoresis with Hydrasys-Hyrys were determined. Within-run imprecision (CV) for fraction quantitation with the semiautomated system was between 1% (albumin) and 4.5% (beta-globulin). Total imprecision (CV) was between 2.7% (albumin) and 7.3% (beta-globulin). Semiautomated agarose electrophoresis showed linear correlation with cellulose acetate electrophoresis. Thirty-four specimens with monoclonal components were analyzed by manual immunoelectrophoresis and immunofixation and by Hydrasys. In one case, a light-chain disease was missed with Hydrasys when the sample was diluted 1:3 (the routine dilution) but not when the sample was assayed undiluted. In another case, the Hydrasys system revealed a small IgGA monoclonal component in addition to the IgA monoclonal component detected by the manual methods. In the other cases, no differences between the manual methods and the semiautomated method were seen with respect to paraprotein identification.

Bence Jones Protein↗