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PubMedDate not supplied

Two-dimensional acrylamide gel electrophoresis of cancer-patient serum proteins.

Sera from normal volunteers, patients with a variety of non-neoplastic diseases and patients with malignant or benign tumors were examined by two-dimensional acrylamide gel electrophoresis. In this technique the serum is first separated in an acrylamide gel column followed by a second electrophoresis at right angles to the first separation in a continuous concave 2 to 30 percent gradient acrylamide gel slab. The stained two-dimensional gel slab appears as a "fingerprint" pattern or "map" of the separated serum proteins. Both qualitative and quantitative differences in the fingerprint patterns of cancer-patient sera were observed. The quantitative alterations did not appear specifically associated with malignant tumors. However, several qualitative differences were detected, of which some may represent markers of malignancy as they were not observed in the normal, abnormal and benign tumors control sera. At least one of the abnormal protein stained spots, a prealbumin, appears to be restricted and related in some way to cancer of the lymphoreticular system. These data, although limited, support earlier indications that two-dimensional acrylamide gel electrophoresis offers a new and powerful tool to the cancer scientist for the detection of alterations in cancer-patient sera. A discussion of its possible use to examine other biological fluid and tumor extracts from the cancer patient is presented.

Blood Protein Electrophoresis↗

PubMed1981

Tumor-associated serum proteins in rats as detected by two-dimensional gel electrophoresis.

The sera from normal and tumor-bearing rats were separated by polyacrylamide gel disc isoelectric electrophoresis and then in a perpendicular dimension by polyacrylamide gel slab electrophoresis with a gradient from 4% to 20%. When the gel was stained with Coomassie brilliant blue, 100-150 components were separately observable with normal serum. With the sera from Rhodamine sarcoma-bearing rats, three more components were detected with a high reproducibility. They were of pI (isoelectric point) 4.5 and Rm (relative mobility) 0.63, of pI4.8 and Rm 0.63 and of pI 4.9 and Rm 0.75. The 4.5/0.63 (pI/Rm) and the 4.8/0.63 components appeared soon after transplanting the tumor, whereas the 4.9/0.75 component appeared at the late stage of its growth. Upon surgical removal of the grown tumor, the former two components disappeared rapidly, while the latter component disappeared gradually. Similar results were obtained with Yoshida sarcoma, and ascites hepatoma AH66 and AH130. A 3rd polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the 4.5/0.63 and the 4.8/0.63 components were each composed of a single polypeptide of approximately 64,000 daltons, and that the 4.9/0.75 component was composed of two different polypeptides of approximately 33,000 daltons and approximately 10,000 daltons. It was confirmed by an immunodiffusion experiment that none of these three proteins was alpha-fetoprotein.

Animals↗

PubMedDate not supplied

Purity testing of recombinant proteins by capillary electrophoresis.

Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties. The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E. coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells. The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies. MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins.

Animals↗

PubMed1998

Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroblasts.

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.

Breast↗

PubMedDate not supplied

Monitoring solution-phase combinatorial library synthesis by capillary electrophoresis.

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations.

Acetamides↗

PubMed1999

High-throughput single-strand conformation polymorphism analysis by automated capillary electrophoresis: robust multiplex analysis and pattern-based identification of allelic variants.

Genetic diagnosis of an inherited disease or cancer often involves analysis for unknown point mutations in several genes; therefore, rapid and automated techniques that can process a large number of samples are needed. We describe a method for high-throughput single-strand conformation polymorphism (SSCP) analysis using automated capillary electrophoresis. The operating temperature of a commercially available capillary electrophoresis instrument (ABI PRISM 310) was expanded by installation of a cheap in-house designed cooling system, thereby allowing us to perform automated SSCP analysis at 14-45 degrees C. We have used the method for detection of point mutations associated with the inherited cardiac disorders long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM). The sensitivity of the method was 100% when 34 different point mutations were analyzed, including two previously unpublished LQTS-associated mutations (F157C in KVLQT1 and G572R in HERG), as well as eight novel normal variants in HERG and MYH7. The analyzed polymerase chain reaction (PCR) fragments ranged in size from 166 to 1,223 bp. Seventeen different sequence contexts were analyzed. Three different electrophoresis temperatures were used to obtain 100% sensitivity. Two mutants could not be detected at temperatures greater than 20 degrees C. The method has a high resolution and good reproducibility and is very robust, making multiplex SSCP analysis and pattern-based identification of known allelic variants as single nucleotide polymorphisms (SNPs) possible. These possibilities, combined with automation and short analysis time, make the method suitable for high-throughput tasks, such as genetic screening.

Alleles↗

PubMed1999

Comparison of radial immunodiffusion and alkaline cellulose acetate electrophoresis for quantitating elevated levels of fetal hemoglobin (HbF): application to evaluating patients with sickle cell disease treated with hydroxyurea.

Radial immunodiffusion (RID), alkaline cellulose acetate electrophoresis, and high-performance liquid chromatography (HPLC) were compared for quantitating the elevated (> 10%) level of fetal hemoglobin (HbF) found in the red blood cells of sickle cell disease patients undergoing treatment with hydroxyurea. HPLC- and electrophoresis-determined values were comparable. The RID-determined values were higher, in many cases twofold higher. False high HbF values would be misleading in assessing the effectiveness of hydroxyurea therapy in sickle cell disease patients. We subsequently initiated an examination of the variation in HbF values due to the use of different HbF radial immunodiffusion QUIPlates and different positions within a single plate in an attempt to determine the cause of these discrepancies. Within-run precision studies indicated that significantly different size precipitin rings were obtained depending upon which area of the plate the hemolysate containing antigen (HbF) was applied. A common feature associated with poor precision plates was a marked difference in degree of coloration of gel throughout the plate. Spuriously high HF concentrations were obtained with antigen (HbF) placed in wells located in the lighter colored gel area while antigen placed in wells in the darker colored area of the agarose gel bed were more in agreement with the electrophoretically determined HbF concentrations. The variation in HbF values was significantly greater in the diluted (HbF QUIPlate Diluent) samples than in the neat samples even on plates of uniform gel coloration. As a result of this study, we will continue to monitor high HbF levels by densitometry following alkaline cellulose acetate electrophoresis.

Adult↗

PubMed1999

Transient association of the DNA-ligand complex during gel electrophoresis.

DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.

Binding Sites↗

PubMed1999

Analysis of apolipoproteins and lipoproteins by capillary electrophoresis.

Capillary electrophoresis provides a valuable analytical tool for the analysis of apolipoproteins and lipoproteins. Sodium dodecyl sulfate (SDS) capillary gel electrophoresis can resolve human and animal apolipoprotein (apo) A-I, apo A-II, apo E and apo A-IV in isolated high density lipoproteins (HDLs) and is capable of analysing HDL apo A-I and apo A-II content with a coefficient of variation (CV) of less than 5%. Capillary zone electrophoresis (CZE) using coated capillaries with Tricine-urea buffer can also be used for the analysis of HDL apolipoproteins and is also capable of resolving apo A-I isoforms, pro-apo A-I, mature apo A-I and deamidated apo A-I. Furthermore it can separate human A-I from endogenous rabbit A-I in transgenic rabbits expressing the human apo A-I gene. Capillary isoelectric focusing can also separate apo A-I isoforms. Analysis of charge-modified low density lipoprotein (LDL) (ox-LDL) produced by in vitro lipid peroxidation can be performed by CZE and the technique can be used to monitor simultaneous changes in the electrophoretic mobility and absorption spectra of LDL.

Animals↗

PubMed1999

Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity.

Single cell gel electrophoresis, also known as the comet assay, is widely used for the detection and measurement of DNA strand breaks. With the addition of a step in which DNA is incubated with specific endonucleases recognising damaged bases, these lesions can be measured, too. In the standard protocol, electrophoresis is carried out at high pH. If, instead, electrophoresis is in neutral buffer, the effect of DNA damage seems to be much reduced--either because alkaline conditions are needed to reveal certain lesions, or because the effect of the same number of breaks on DNA migration is greater at high pH. A lower sensitivity can be useful in some circumstances, as it extends the range of DNA damage levels over which the assay can be used. Here we compare the performance of standard and modified techniques with a variety of DNA-damaging agents and offer possible explanations for the differences in behaviour of DNA under alternative electrophoretic conditions.

Buffers↗

PubMed1999

Monitoring nitrotyrosinylation of a synthetic peptide by capillary zone electrophoresis.

Proteins may be nitrated on tyrosyl residues (nitrotyrosinylated) by the action of reactive nitrogen species in inflamed tissues. Capillary electrophoresis was used to monitor this reaction in a model system with tetranitromethane as the nitrotyrosinylating reagent and a synthetic pentapeptide containing one tyrosine as the target molecule. The reaction was readily followed by capillary electrophoresis performed at pH 8 and, using an absorption wavelength of 436 nm, the signature spectral characteristics of the nitrotyrosinylated peptide were verified on-line. The peak appearance time for the nitrotyrosinylated peptide was more than 1 min longer than that of the starting material and a single main product was observed in contrast to the case when peroxynitrite was used as the nitrotyrosinylating reagent. Capillary electrophoresis appears to be a convenient method for the optimization of nitrotyrosinylation, examination of reaction inhibitors, and for studies of the consequences of nitrotyrosinylation, e.g., for antibody binding and for the function of the target protein or peptide.

Electrophoresis, Capillary↗

PubMed1999

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.

Animals↗

PubMed1999

Detection of point mutations by capillary electrophoresis with temporal temperature gradients.

By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.

Animals↗

PubMed1999

Comparison between transient isotachophoretic capillary zone electrophoresis and reversed-phase liquid chromatography for the determination of peptides in plasma.

Low levels of peptide drugs in human plasma can be determined employing off-line solid-phase extraction, followed by capillary zone electrophoresis with UV detection. A bioanalytical procedure is presented, using gonadorelin and angiotensin II in human plasma as model compounds. The solid-phase extraction method, based on a weak cation exchange mechanism, is able to remove interfering endogenous components from the plasma sample, extract the model peptides quantitatively, and give a possibility of concentrating the sample at the same time. Transient isotachophoretic conditions were kept to increase the sample loadability by about two orders of magnitude. Up to about 70% of the capillary was filled with the reconstituted extract, whereafter the peptides were selectively concentrated during the first 15 min. Subsequently, the concentrated sample zones were separated under capillary zone electrophoresis conditions, showing the technique's high resolution. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility was observed in the 20-100 ng/mL concentration range. A more extensive washing procedure permits quantitation of gonadorelin at the 5 ng/mL level. In comparison with a liquid chromatography analysis, superior mass sensitivity and separation are obtained with the transient isotachophoretic capillary zone electrophoresis method. Moreover, in this case equivalent sensitivity is achieved when it is directly compared with a liquid chromatography method with UV detection, keeping in mind that 60 times more sample is needed for the latter method. A further gain in sensitivity can be obtained when the analysis is combined with native fluorescence detection, as is demonstrated by combining liquid chromatography separation with fluorescence detection.

Chromatography, Liquid↗

PubMed1999

Capillary electrophoresis of peptides.

This article gives a review of the recent developments in capillary electrophoresis (CE) of peptides. New approaches to the theoretical description of electromigration behavior of peptides are described, and methodological aspects of CE separations of peptides such as selection of separation conditions, sample treatment, suppression of peptide adsorption to the capillary wall and specificities of CE separation modes are discussed. Progress in application of high performance detection schemes, namely laser-induced fluorescence and mass spectrometry, in peptide separations by CE is presented. Applications of different CE techniques, zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography to peptide analysis, preparation and physicochemical characterization are demonstrated.

Electrophoresis, Capillary↗

PubMed2000

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries.

The methodology and instrumentation for fast denaturing electrophoresis in short capillaries was developed and exemplified by detection of short tandem repeat polymorphism in the endothelin 1 gene. The resolution of two nucleotides, which is required for the detection of a dinucleotide repeat polymorphism, was achieved in a capillary of an effective length of 2.5 cm at a temperature of 600C and an electric field strength of 600 V/cm in 42 s. Thus, the use of denaturing electrophoresis in short capillaries with laser-induced fluorescence detection resulted in a reduction of analysis time by a factor of 200 when compared to the conventional slab gel electrophoresis. The developed methodology and instrumentation is advantageous for an implementation in clinical diagnostics and genetic population screening where fast analytical instrumentation amenable to automation is of paramount importance.

DNA↗

PubMed2000

Applications of capillary electrophoresis with electrochemical detection in pharmaceutical and biomedical analyses.

As a high efficiency separation technique, capillary electrophoresis has been widely used in various fields of analytical science. This review discusses the applications of electrochemical detection systems combined with capillary electrophoresis in pharmaceutical and biomedical analysis. These detection methods mainly involve amperometric detection but also include conductivity detection and potentiometric detection. Its applications in the field are divided into six parts, including catechol compounds, thiols, amino acids and peptides, carbohydrates, general pharmaceuticals, and other related compounds. A relatively detailed discussion is described for each compound under the current studied. On this basis, we have suggested several conceivable directions for capillary electrophoresis with electrochemical detection in the future.

Electrochemistry↗

PubMed2001

Multidimensional high performance liquid chromatography--capillary electrophoresis separation of a protein digest: an update.

The trypsin digest of a mixture of two proteins, namely cytochrome c and myoglobin, was first separated in the first dimension by high-performance liquid chromatography (HPLC). Fractions from the HPLC were collected every 30s with the aid of a fraction collector into a 96-well microtiter plate. After concentration, all the collected fractions were analyzed simultaneaosly in the second dimension by a 96-array capillary electrophoresis system. The labeled peptides were detected by laser-induced fluorescence. An internal standard, allura red, was added to all the fractions, prior to capillary electrophoretic analysis. The internal standard serves two functions, migration time correction and signal intensity correction. The data are presented in two different formats, as an electropherogram of all the fractions and in a two-dimensional (2-D) format. The 2-D plot of the data shows the density of each spot, which corresponds to the concentration of the migrating peptides. The total experimental time for the HPLC and capillary electrophoretic analyses ist less than 1 h, which ist much faster than using 2-D slab-gel electrophoresis or single-capillary capillary electrophoresis.

Chromatography, High Pressure Liquid↗