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PubMed1975

A micro-method for the assay of polyadenylate-containing ribonucleic acid by gel electrophoresis.

A micro-method for the determination of the electrophoretic profile of the various poly(A)-containing RNA species in a RNA sample was developed. The method is simple to carry out and allows for great reproducibility. It combines the resolving power of electrophoresis in agarose with the specificity of binding of poly(A) to poly(U)-containing glass-fibre filters. It consists of the following steps. (1) The molecules in an RNA sample are first separated according to their molecular weight by electrophoresis in agarose, at low ionic strength. 2. The molecules thus seperated are then submitted to a second electrophoresis in 'binding buffer' in a direction perpendicular to the first one. In the course of this electrophoresis the poly(A)-containing RNA species are seperated from other RNA species as they bind to a poly(U)-containing glass-fibre filter which is placed across the electrophoresis path. The method was used to determine the electrophoretic profile of Rhynchosciara salivary-gland messenger RNA. It was found that the population of messenger RNA in the gland is dominated by forms moving as 18 and 158 S peaks, but there is also polydisperse RNA with slower mobility.

Animals↗

PubMed1999

Comparison of capillary zone and immunosubtraction with agarose gel and immunofixation electrophoresis for detecting and identifying monoclonal gammopathies.

Capillary zone electrophoresis (CZE) and immuno-subtraction electrophoresis (ISE) were evaluated for ability to detect and immunotype monoclonal proteins, compared with agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE), respectively. Six hundred seventeen serum samples were analyzed with CZE and AGE to determine sensitivity and specificity in detecting IFE-confirmed monoclonal gammopathies. Both techniques detected all monoclonal spikes due to IgM (n = 8), IgG (n = 38), and free light chains (n = 3). Agarose gel electrophoresis, however, detected only 11 of 14 (79%) IgA monoclonal spikes detected with CZE. In a second study, 78 serum samples, 48 of which had a monoclonal gammopathy confirmed with IFE, were evaluated with ISE. Only 60% to 75% of the monoclonal gammopathies were correctly immunotyped with ISE by 4 readers blinded to the IFE immunotype. Thus CZE was more sensitive than AGE in detecting low concentrations of monoclonal proteins, but ISE is less accurate than IFE in determining the immunotype of the monoclonal gammopathy.

Autoanalysis↗

PubMedDate not supplied

Is cellulose acetate electrophoresis a suitable technique for detection of Hb Bart's at birth?

Symptomatic alpha-thalassemia (alpha-thal) as found in South-East Asia is uncommon in India. However, the presence of Hb Bart's in cord blood samples has been reported from different parts of India and the prevalence of alpha-thal has ranged from 0.5 to 18% by different electrophoretic techniques. The methodology utilised has ranged from paper electrophoresis to isoelectric focussing (IEF). We screened 798 cord bloods for the presence of Hb Bart's by cellulose acetate electrophoresis and found a prevalence rate of alpha-thal of 15.3% in a heterogenous population in Bombay. A comparison of four different electrophoretic techniques for detection of Hb Bart's in 138 neonates showed that cellulose acetate and starch gel electrophoresis were by and large comparable and only a little less sensitive than IEF. Paper electrophoresis used at many centers in India was most insensitive. As alpha-genotyping is not possible at most centers in the country, it is suggested that a simple cellulose acetate electrophoresis would be the method of choice for screening neonates for alpha-thal in India. As a part of our follow-up study, alpha-genotyping was done by Southern blot hybridization in 24 cases who had shown variable levels of Hb Bart's at birth. The rightward deletion (-alpha3.7/) either in a heterozygous or homozygous condition was the only gene defect encountered in this preliminary study. However, 7 of 24 cases (29.17%) showed no correlation between Hb Bart's level and alpha-genotypes.

Electrophoresis↗

PubMed1979

Separation of two serum very low density lipoprotein fractions using starch block electrophoresis.

A quantitative electrophoretic method has been developed in order to differentiate very low density (VLDL) pre-beta lipoproteins from late pre-beta lipoproteins using starch as a supporting medium. It was possible to obtain a bimodal distribution of lipoprotein lipids from VLDL which on agarose gel electrophoresis had a pre-beta band and a late pre-beta band. Optimal conditions were: ammonium carbonate buffer, mu = 0.025, dialysis prior to electrophoresis. Agarose gel electrophoresis demonstrated that the fast and slow components obtained on starch block electrophoresis corresponded to the pre-beta and late pre-beta band respectively. With increasing migration towards the anode the ratio of cholesterol to triglycerides decreased continuously. It is suggested that the fast triglyceride rich component represent newly secreted VLDL species and the slower component mainly postlipolytic particles. The pre-beta band on agarose gel electrophoresis might represent more newly secreted VLDL than the late pre-beta band. However, it cannot be excluded that part of late pre-beta lipoproteins may be secreted de novo.

Blood Proteins↗

PubMed1999

[Capillary free zone electrophoresis of globin chains].

OBJECTIVE: To introduce a new technique for rapid separation of globin chains from clinical samples. METHODS: The authors used the uncoated capillary and the electrophoresis buffer which contained 100mmol/L NaH2PO4 +7mmol/L urea +1% Triton X-100 +1% 2-mercaptoethanol +0.25% PEG-600. Capillary free zone electrophoresis was employed in analyzing the hemoglobin-water solutions which were made from HbA control and 74 clinical blood samples. The globin chains were separated from the samples. RESULTS: IN 40 minutes, five globin chains: alpha, beta, Ggamma, delta, Agamma were separated clearly by capillary free zone electrophoresis using nanolitres of sample. In electrophoresis figure the baselines were stable and the peaks were clear. The time coefficient of variation (CV) was under 3%, and the area CV below 4%. Also, The authors measured the relative concentration of globin chains from 60 normal cases and 14 thalassemia patients. CONCLUSION: The capillary free zone electrophoresis for the separation of globin chains from clinical samples has been successfully achieved. It is a rapid, simple and sensitive technique suitable for rapid diagnosis of hemoglobinopathies.

Adult↗

PubMed1999

Plastic microchip electrophoresis for analysis of PCR products of hepatitis C virus.

BACKGROUND: Electrophoresis on polymeric rather than glass microstructures is a promising separation method for analytical chemistry. Assays on such devices need to be explored to allow assessment of their utility for the clinical laboratory. METHODS: We compared capillary and plastic microchip electrophoresis for clinical post-PCR analysis of hepatitis C virus (HCV). For capillary electrophoresis (CE), we used a separation medium composed of 10 g/L hydroxypropyl methyl cellulose in Tris-borate-EDTA buffer and 10 micromol/L intercalating dye. For microchip electrophoresis, the HCV assay established on the fused silica tubing was transferred to the untreated polymethylmethacrylate microchip with minimum modifications. RESULTS: CE resolved the 145-bp amplicon of HCV in 15 min. The confidence interval of the migration time was <3.2%. The same HCV amplicon was resolved by microchip electrophoresis in <1.5 min with the confidence interval of the migration time <1.3%. CONCLUSION: The polymer microchip, with advantages that include fast processing time, simple operation, and disposable use, holds great potential for clinical analysis.

DNA, Viral↗

PubMedDate not supplied

Atypical findings of protein studies using capillary zone electrophoresis.

We present two atypical cases of protein studies that were evaluated by immunofixation and immunosubtraction using capillary zone electrophoresis and high-resolution agarose gel electrophoresis. The first study showed an abnormal peak in the beta region by capillary zone electrophoresis that was located in the gamma region of the high-resolution agarose gel electrophoresis. Further investigation showed that this monoclonal protein was displaced due to binding with beta-lipoproteins. In the second case, a large peak was detected in the alpha-2 region and was shown by capillary zone electrophoresis to be a non-proteinaceous material that mimicked a paraprotein.

Aged↗

PubMed2001

Computer-supported detection of M-components and evaluation of immunoglobulins after capillary electrophoresis.

BACKGROUND: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. METHODS: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. RESULTS: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. CONCLUSIONS: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

Aged↗

PubMed1991

Capillary electrophoresis--a new clinical tool.

The potential clinical diagnostic utility of capillary electrophoresis in an open-tubular column is established. Separation patterns for serum proteins by conventional agarose gel electrophoresis can be faithfully reproduced by a capillary electrophoresis procedure that provides the complete run data in 8 min. Hemoglobin variants can be separated within 10 min. The capillary electrophoresis separations are performed reliably and reproducibly in an untreated 75 micron (i.d.) x 25 cm fused-silica column. Diluted serum or hemoglobin samples can be loaded on an automated instrument with on-line injection, detection, and quantification, providing a truly "walkaway" electrophoresis system.

Blood Proteins↗

PubMed1989

A hybrid mode of rotating gel electrophoresis for separating linear and circular duplex DNA.

During agarose gel electrophoresis, alternately applying the electrical potential gradient (E) in two directions (separated by an angle, psi) has been used to enhance the separation by length of linear, double-stranded DNA; the value of psi is usually between 0.5 pi and 0.7 pi radians (Cantor et al. (1988). Ann. Rev. Biophys. Biophys. Chem. 17, 287-304). When the direction of E is changed by rotating the gel (rotating gel electrophoresis, or RGE), open circular DNA longer than 48 Kb is found here to form bands that decrease in either sharpness or migration distance as psi is decreased from 1.1 pi to 0.5 pi radians. A satisfactory band is formed only for psi greater than or equal to 1.1 pi radians. In contrast, the previously-demonstrated separation and banding of linear DNAs (48.5-800 Kb) was observed for psi values between 0.6 pi and 1.4 pi radians. The cause of psi's effect on open circular DNA is electrophoresis during the gel's rotation. RGE by use of psi values above 1.0 pi is a hybrid mode of RGE, incorporating favorable characteristics of both the usual mode and a more recently demonstrated (Serwer, P. and Hayes, S.J. (1989). Applied and Theoretical Electrophoresis, 1, 95-98) mode in which psi is 2 pi radians. To separate linear from open circular DNA and to fractionate by length both of these conformational forms, the following procedure of two-dimensional agarose gel electrophoresis has been developed: (a) a first dimension performed by use of RGE and a psi of 1.4 pi radians, (b) a second dimension performed with an invariant electrical field. Circular DNA has been found among the bacteriophage lambda DNA concatemers (48.5-800 Kb) used as length standards. By use of a psi = 1.4 pi radians, the band of 48.5 Kb monomeric open circular lambda DNA is sometimes accompanied by at least three other bands of open circular DNA; the latter are presumed to be those formed by dimeric, trimeric and tetrameric open circular lambda DNA.

Bacteriophage lambda↗

PubMed1995

DNAOPT: a computer program to aid optimization of DNA gel electrophoresis and SDS-PAGE.

Several methods and computer programs have been developed for estimating the size of DNA fragments from gel electrophoresis. However, methods are lacking that may facilitate in optimization of gel conditions. In this article, a computer program called DNAOPT is described, which was developed to assist researchers in tuning the gel conditions of gel electrophoresis. The DNAOPT program fits the reciprocal of the migration distance vs. the size of the DNA fragments using the hyperbolic regression method and computes the hyperbolic parameters such as signal, flatness and capacity (optimization parameters). The program further manipulates these parameters obtained by running gel electrophoresis under various conditions (i) to determine the relationship between the gel conditions (temperature, buffer concentration, electric field strength, etc.) and optimization parameters; (ii) to demonstrate gel electrophoresis curves and optimization parameters graphically; and (iii) to represent the optimizing parameters at different gel conditions in tabular form. The above-mentioned program options aid the users in selecting optimum gel conditions by running the gel repeatedly under various conditions in which the agarose concentration, electric field strength, temperature, buffer concentration and so on are varied in a systematic way for each set of gel conditions. Similarly, this program can also be used to optimize gel conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Algorithms↗

PubMed1978

[The role of various methods of electrophoresis in the diagnosis of paraproteinemia].

For the estimation of different electrophoresis methods for indication and identification of paraproteins as routine methods are proposed the cellulose acetate electrophoresis and the fixation immunoelectrophoresis in cellulose acetate foils. Under adequate conditions the agar gel electrophoresis working with larger separability can be used together with immunochemical investigations. In examinations of uroproteins and also for the identification of rare and atypical cases of paraproteinaemia (narrower and several M-gradients, immunoglobulin fragments and complexes) in connection with the methods mentioned also the electrophoresis in the polyacrylamide gel (PAG and in the system PAG-SDS) can represent a valuable information.

Antigen-Antibody Complex↗

PubMedDate not supplied

A nondenaturing protein map of human plasma proteins correlated with a denaturing polypeptide map combining techniques of micro two-dimensional gel electrophoresis.

Human plasma proteins were separated by combining four types of two-dimensional electrophoresis (2-DE) techniques to obtain systematic information on proteins and their constituent polypeptides. A micro gel system was employed to facilitate the analysis. A plasma sample was first analyzed under nondenaturing conditions of electrophoresis (Type I 2-DE) to characterize the properties of proteins under physiological conditions. The sample was then analyzed, employing nondenaturing isoelectric focusing in the first dimension and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension (Type II 2-DE), to study the dissociation of noncovalently bound protein subunits. In the third type of 2-DE (Type III 2-DE), proteins were separated by nondenaturing isoelectric focusing and treated with urea/mercaptoethanol/SDS and then subjected to second-dimension SDS electrophoresis, to study the dissociation of disulfide-bonded polypeptides. In the fourth type of 2-DE (Type IV 2-DE), the conditions of denaturing 2-DE were employed; the sample was treated with SDS-mercaptoethanol-urea-Nonidet P-40, separated by denaturing isoelectric focusing, and then subjected to SDS electrophoresis. The combined 2-DE technique will be useful to construct a comprehensive database of plasma proteins combining a "nondenaturing protein map" (a protein map) and a "denaturing protein map" (a polypeptide map).

Adult↗

PubMed2000

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis.

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.

Animals↗

PubMed2001

Improving the length-fractionation of DNA during capillary electrophoresis.

The present study develops a path-lengthening strategy for capillary electrophoresis of short double-stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two-stage cycles (cyclic electrophoresis): The first analysis-stage of each cycle is a constant field (forward) capillary electrophoresis. This analysis-stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement-stage of each cycle is zero-integrated field electrophoresis (ZIFE). The enhancement-stage improves the DNA length-fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement-stage. Increase in both peak separation and peak sharpness contribute to improvement in the length-fractionation of DNA molecules.

DNA↗

PubMed2002

Applications of on-line weak affinity interactions in free solution capillary electrophoresis.

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.

Animals↗

PubMed1991

Shielding effects of small ions in gel electrophoresis of DNA.

Results of free electrophoresis and free sedimentation of charged rods are introduced to allow for electrical double-layer effects in the gel electrophoresis of DNA, modeled as a random flight sequence of rodlike Kuhn chain elements. The kinetics of DNA is shown to depend strongly on the orientation of the chain elements with respect to the external field and with respect to the direction of their velocity. The new theory gives approximate agreement between the DNA charge derived from electrophoresis of free and tethered DNA (close to 1.0e per DNA phosphate in both cases), compared with the earlier discrepancy of a factor 10 or 20 for the theory without shielding by salt. As part of a realistic force field for macromolecular dynamics simulation, the small ion effects are included in an expression for the gel forces restraining the DNA in gel electrophoresis from moving as in free electrophoresis.

DNA↗

PubMed1994

Pulsed-field gel electrophoresis for the separation of large protein molecules exemplified by the isoforms of apolipoprotein (a).

The performance of pulsed-field gel electrophoresis (PFGE) was assessed for the separation of protein molecules. The allelic isoforms of apolipoprotein (a) (apo[a]) served as a model for this study because apo(a) is an unusually large protein, consisting of a variable number of repeating units. PFGE and, for comparison, conventional electrophoresis of human sera were carried out under reducing conditions in agarose gel. After blotting proteins onto nitrocellulose membrane, a combination of monospecific rabbit anti-apo(a) antibody and alkaline phosphatase-conjugated protein A detected apo(a) isoforms in all sera tested. The various apo(a) isoforms were effectively resolved within two repeating units ("kringles") by both PFGE and conventional electrophoresis, but the type of agarose gel used greatly affected the speed of electrophoretic separation. In a series of 89 human sera, 59 double-band and 30 single-band patterns were seen using either electrophoretic system. However, one specimen produced double bands with PFGE, while only a single band could be detected by conventional electrophoresis, and with another specimen the opposite occurred. A total of 22 different apo(a) isoforms were identified among these patterns. It is concluded that the increasingly available PFGE technology is a practical alternative to conventional agarose electrophoresis for the separation of large protein molecules.

Apolipoproteins A↗