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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↗
PubMed2004

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis.

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.

Electrophoresis, Agar Gel↗
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↗
PubMed1989

A new mode of rotating gel electrophoresis for fractionating linear and circular duplex DNA: the effects of electrophoresis during the gel's rotation.

Linear and circular DNA have been fractionated by use of rotating gel electrophoresis (RGE; RGE is a form of pulsed-field gel [PFG] electrophoresis) in a mode not previously described. This new mode consists of electrophoresis while repeating the following two-step process: rotating the gel by 2 pi radians, followed by holding the gel motionless. With this new mode of RGE: (1) The fractionation by length of linear DNA is greater than that achieved without changing the direction of the field. (2) The simultaneous separation of open circular DNA (48.5-97 Kb) from linear DNA (1-180 Kb) and fractionation by length of open circular DNA is achieved. These latter separations have not previously been achieved by a one-dimensional electrophoresis. The mechanism of linear DNA's sieving in the new mode appears different from that of all previously described procedures of PGF electrophoresis.

Bacteriophage lambda↗
PubMed2000

A decade of capillary electrophoresis.

Since the introduction of the first commercial capillary electrophoresis (CE) instrument a decade ago, CE applications have become widespread. Today, CE is a versatile analytical technique which is successfully used for the separation of small ions, neutral molecules, and large biomolecules and for the study of physicochemical parameters. It is being utilized in widely different fields, such as analytical chemistry, forensic chemistry, clinical chemistry, organic chemistry, natural products, pharmaceutical industry, chiral separations, molecular biology, and others. It is not only used as a separation technique but to answer physicochemical questions. In this review, we will discuss different modes of CE such as capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, capillary isoelectric focusing, and capillary electrochromatography, and will comment on the future direction of CE, including array capillary electrophoresis and array microchip separations.

Amino Acids↗
PubMedDate not supplied

Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate-electrophoresis method and rapid two-dimensional electrophoresis.

Two methods suitable for routine clinical analyses of urinary proteins are presented and compared. The first is a horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique, suitable for simultaneous analysis of 20 native urinary samples. This method uses polyacrylamide gradient gels, prepared with a laboratory-built gel casting device. The second method is a rapid two-dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate-electrophoresis. The first step uses a routine system (Chemetron), the second separation step followed by staining with Coomassie Brilliant Blue R is performed on the PhastSystem. The resulting two-dimensional patterns reveal urinary proteins distributed according to the 5-zone pattern of native proteins (albumin, alpha-1, alpha-2, beta, gamma-globulin) as well as to the logarithm of their molecular weights. Examples of (routine) diagnoses with a special interest in the monitoring of kidney transplant patients are shown.

Cellulose↗
PubMed1990

Scale-up of free flow electrophoresis: I. Purification of alcohol dehydrogenase from a crude yeast extract by zone electrophoresis.

The potential and limitations in scaling-up free-flow electrophoresis, with emphasis on zone electrophoresis, are demonstrated. Purification of alcohol dehydrogenase (ADH) from a crude yeast extract was chosen as a model for an industrial approach to enzyme purification. In zone electrophoresis the separation quality strongly depends on the pH and conductivity of the background electrolyte, its residence time and flow rate, as well as the applied voltage. Optimization of these parameters resulted in a purification factor of 5.3 and a yield of 96% ADH, using a Tris/HCl buffer, pH 8.0, and a conductivity of 1 mS/cm, with a residence time of 10 min at 500 V. The loading capacity of the method for a laboratory-sized free-flow electrophoresis apparatus was limited to a sample throughput of about 0.4 g/h. By increasing the chamber dimensions it was possible to purify the enzyme by a purification factor of 4.7 and a yield of 93% ADH, at a throughput of about 1 g total protein/h. By simultaneously applying the sample at 3 input positions the throughput could be increased to 2.75 g/h with a purification factor of 4.7 and an overall yield of 90%.

Alcohol Dehydrogenase↗
PubMed1996

Distinction between genotypes of Lupinus species by sodium dodecyl sulphate-gel electrophoresis and by capillary electrophoresis.

Effective distinction was achieved among a wide range of lupin grain samples by either sodium dodecyl sulphate (SDS)-gel electrophoresis or capillary electrophoresis, based on grain-protein composition. Capillary electrophoresis was faster (< 1 h) and provided slightly greater distinction between the samples. On the other hand, SDS-gel electrophoresis could provide a greater through-put of samples in a 24 h period. Either technique could be used successfully to distinguish between lupin species and cultivars for taxonomic analysis or seed identification.

Electrophoresis, Capillary↗
PubMed1997

Comparison of serum protein electrophoresis by agarose gel and capillary zone electrophoresis in a clinical setting.

Capillary zone electrophoresis (CZE) offers the potential for automating serum protein electrophoretic analysis traditionally performed on standard thin-layer agarose gels. The following describes the use of CZE compared to agarose gel electrophoresis (AGE) for the detection of dysproteinemia and paraproteinemia in a clinical study involving 240 patients. The study includes within-run and between-run reproducibility data on the Paragon CZE 2000 Clinical Capillary Electrophoresis System, in addition to concordance data between the two methodologies. Paraprotein quantitation studies comparing AGE versus CZE were also performed. Reproducibility for the automated CZE system was superior to the AGE system. Improved reproducibility for the CZE method is largely due to measuring protein absorbance directly at 214 nm versus the traditional AGE method that measures the amount of dye adsorbed to protein. Reproducibility data as percent coefficient of variance (% CV) for the five classic bands in a normal control serum for between-run precision ranged from 1.2 to 4.5% for CZE compared to AGE, which ranged from 3.8 to 8.0% CV. Concordance studies between AGE and CZE involving dysproteinemias including hypogammaglobulinemia, polyclonal and monoclonal gammopathies, acute and chronic inflammation, nephrosis, hepatodegenerative disease, cirrhosis, and iron deficiency anemia showed 96% agreement. Paraprotein classification, which compared the CZE immunosubtraction method to immunofixation electrophoresis (IFE) on agarose, showed 100% agreement. Certain dysproteinemias involving beta lipoprotein were in partial concordance due to the inability of the CZE procedure to detect this component. Detection limits for monoclonal gammopathies, providing they were not comigrating with other proteins, were IgG 50 mg/dL, IgM 75 mg/dL, and IgA 75 mg/dL. Paraprotein quantitative studies between the two methods showed less than a +/- 0.2 g/dL variation.

Blood Proteins↗
PubMed1998

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis.

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.

Blood Proteins↗
PubMed2005

Microchip analysis of lithium in blood using moving boundary electrophoresis and zone electrophoresis.

The determination of inorganic cations in blood plasma is demonstrated using a combination of moving boundary electrophoresis (MBE) and zone electrophoresis. The sample loading performed under MBE conditions is studied with the focus on the quantitative analysis of lithium. A concentration adjustment takes place when the sample components migrate into the chip during the sample loading step. Using a heart-cutting method, a diluted sample plug is subsequently separated with capillary zone electrophoresis. The excessive dispersion that is typical of the samples with a high ionic strength is thereby prevented. The method can be easily applied to commercially available capillary electrophoresis microchips under the condition that the electroosmotic flow is suppressed. For the first time the lithium concentration is determined in the blood plasma from a patient on lithium therapy without sample pretreatment. Using a microchip with conductivity detection, a detection limit of 0.1 mmol/L is obtained for lithium in a 140 mmol/L sodium matrix.

Electric Conductivity↗
PubMed1979

Diagnostic value of brief microzone electrophoresis of unconcentrated CSF and agar gel electrophoresis of concentrated and unconcentrated CSF.

This paper reports a comparison between brief microzone electrophoresis and agar gel electrophoresis of CSF proteins; 41 different CSFs were examined simultaneously by all three methods. Fractionation of gamma-globulins was found by brief microzone electrophoresis in 53.7% of unconcentrated CSF. This fractionation was found in 63.4% of unconcentrated CSF after agar gel electrophoresis, in 48.8% of concentrated CSF. Serum-like pherogram and the rapid alpha-globulin type were found equally often.

Cerebrospinal Fluid Proteins↗
PubMed1995

Evaluation of zone electrophoresis of serum proteins performed on the Helena Laboratories rapid electrophoresis analyser.

Zone electrophoresis of serum proteins is still widely performed as a routine procedure in clinical laboratories. It is used in the diagnosis and management of many disorders, e.g. monoclonal gammopathies, cirrhosis, nephrotic syndrome, acute-phase reaction, immunoglobulin deficiency and others. The aim of this work is to evaluate the analytical performance of zone electrophoresis of serum proteins carried out in the Helena Laboratories Rapid Electrophoresis analyser (REP) comparing the results with those obtained in the Olympus Hite System 200 (HS-200). The REP system employs agarose gel as support medium, the HS-200 system employs cellulose acetate and it is the routine method in our laboratory. To date, we have not found any paper that deals with the assessment of a system for performing zone electrophoresis of serum proteins in terms of comparison with another.

Adolescent↗
PubMed1998

Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system.

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.

Acute-Phase Reaction↗
PubMed1988

Peptide mapping of polypeptides separated by two-dimensional electrophoresis: protease digestion directly on the two-dimensional gel followed by electrophoresis in reverse direction.

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two-dimensional electrophoresis. After two-dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be visualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: comparison of mutation detection enhancement-polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels.

Cutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex-loaded polyacrylamide gel electrophoresis on MDE (mutation detection enhancement) gels (HD-MDE PAGE), of heteroduplex-loaded temperature gradient gel electrophoresis (HD-TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin-fixed, paraffin-embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD-MDE PAGE in 22, by HD-TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD-MDE PAGE and HD-TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD-TGGE and FA detected a clonal pattern down to a dilution of 10(3) monoclonal cells in 10(6) peripheral blood mononuclear cells (PBMC), while HD-MDE PAGE revealed a detection limit of 10(4) monoclonal cells in 10(6) PBMC. In conclusion, HD-TGGE and FA possess a higher sensitivity and lower detection limit than HD-MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD-TGGE.

Cell Separation↗