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PubMed1987

Horizontal two-dimensional electrophoresis of plasma butyrylcholinesterase.

Genetic variants of human plasma butyrylcholinesterase have been characterized and are highly relevant to anesthesiology. They might also represent potential genetic markers for neuropsychiatric disorders. Two-dimensional electrophoresis with isoelectrofocusing in the first and polyacrylamide gel electrophoresis in the second dimension has proved to be a powerful tool in search for genetic variants. Butyrylcholinesterase is an oligomeric enzyme with considerable charge heterogeneity. Conventional two-dimensional electrophoresis proved unsuitable for this enzyme possibly due to its tendency to aggregate by hydrophobic interactions. The inversion of the sequence applying polyacrylamide gel electrophoresis in the first and isoelectric focusing in the second dimension circumvented this problem.

Butyrylcholinesterase↗

PubMed1998

Optimized detection of DNA point mutations by double gradient denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis displays the highest detection rate among mutation scanning methods. In classical denaturing gradient gel electrophoresis the denaturant gradient range and migration times vary for every amplicon to be scanned, greatly affecting the routine application of the method. As an alternative, we developed double gradient denaturing gradient gel electrophoresis where a gradient of pore size is superimposed over the denaturing one, allowing maintenance of the zone-sharpening effect even over prolonged time runs, and adoption of identical run time conditions for all fragments analyzed. Here double gradient denaturing gradient gel electrophoresis has been applied to the analysis of a number of point mutations and polymorphisms located in several exons of three different genes, the cystic fibrosis transmembrane conductance regulator, the beta-globin and the p53 genes.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed2001

Routine differential diagnosis of proteinurias by capillary electrophoresis.

Capillary electrophoresis is a relatively new analytical technique that begins to have an impact on both routine and research in clinical laboratories. Recently, a fully automated system has become commercially available (Paragon CZE 2000, Beckman, USA) for the analysis of human serum proteins. Urine protein analysis, on the other hand, is currently accomplished by electrophoresis of concentrated urine specimens. The method is used to distinguish the glomerular from the tubular proteinuria and for the identification of Bence-Jones proteins. The procedure is labor-intensive and technically demanding. We developed a technique for the serum capillary electrophoresis instrument that can also be applied routinely to the differential diagnosis of proteinurias. Overriding the programmed dilution step of the instrument, we were able to distinguish different types of proteinurias without concentration of specimens with a total protein content of 150-200 mg/l as determined by sulfosalicylic acid. The different electrophoretic patterns obtained by the capillary electrophoresis system for various specimens correlated well with established techniques (Hydragel Proteinurie Kit, Sebia, France). The method is applicable for routine analysis of urinary proteins. It is reliable, less expensive and faster than the conventional methods (electrophoretic or immunonephelometric) used today for the differentiation of proteinurias, and it can be used as a quick screening test.

Albuminuria↗

PubMed2004

Simplified method for the diameter sizing of serum low-density lipoprotein using polyacrylamide gradient gel electrophoresis.

The appearance of small, dense, low-density lipoprotein in serum has been demonstrated to be associated with increased risk of coronary artery disease. The molecular diameter of low-density lipoprotein is usually measured on the basis of mobility differences on polyacrylamide gel electrophoresis. However, since mobility assessed by this method is seriously affected by the increased levels of serum free fatty acids associated with hypertriglyceridemia, we used polyacrylamide gradient gel electrophoresis to eliminate the interference by fatty acids and devised a simple, precise method of polyacrylamide gradient gel electrophoresis to measure the diameter of small, dense, low-density lipoproteins in serum. We used apoferritin and thyroglobulin, which have a molecular diameter of 12.2 nm and 17.0 nm, respectively, and standard low-density lipoprotein particles having a diameter of 25.7 and 27.0 nm as calibrators, estimated by measurement of negative staining of electron microscopy. We also included apoferritin as an internal standard for polyacrylamide gradient gel electrophoresis. The only stain used was Coomassie brilliant blue, and it was used for lipoprotein staining. When we used low-density lipoprotein of 25.73 nm in diameter as a quality control specimen, the coefficient of variation of the size measurements obtained by our method was less than 1.2%. The new method markedly improved the laboratory procedure for measuring the diameter of low-density lipoproteins.

Apoferritins↗

PubMed2001

Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations.

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.

Autoanalysis↗

PubMed2002

Detection of mutations in exon 8 of TP53 by temperature gradient 96-capillary array electrophoresis.

Various capillary electrophoresis applications have increasingly been utilized in mutation detection. Separation of two species is either based on secondary structure or differences in melting of DNA due to the mutation. Detection of the mutant is based on its mobility difference in the sieving matrix. We have adapted a regular 96-capillary sequencing instrument, the MegaBACE 1000, for mutation detection based on thermodynamic stability and mobility shift during electrophoresis. Denaturation of the lower melting domain of the DNA was achieved with a gradually decreasing temperature gradient in combination with a chemical denaturant. Samples were analyzed for mutants in exon 8 of the TP53 genefrom tumor samples and controls. Genomic DNA was PCR-amplified with one fluorescein labeled primer and one GC-clamped primer, diluted in water, and analyzed by temperature gradient 96-capillary array electrophoresis. Tumor samples and PCR reconstruction experiment samples were resolved by capillary gel electrophoresis under appropriate temperature gradient denaturing conditions. Ninety-six samples were analyzed in one run, with an analysis time of 30 min and a sensitivity to detect mutated alleles in wild-type background down to 0.4%. The technique proved to be robust, in that the gradient compensatesfor temperature differences within the capillary chamber; thus, each capillary will pass through the optimal separating conditions around the theoretical melting temperature for TP53 exon 8, separating homoduplexes and heteroduplexes. This technique is applicable to any sequence previously analyzed by DNA melting gel techniques or sequences harboring iso-melting domains of 100-120 bp.

Colorectal Neoplasms↗

PubMed1996

Temperature-programmed capillary electrophoresis for detection of DNA point mutations.

A method for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is reported here. The method is based on the principle of temperature gradient gel electrophoresis (TGGE), a variant of denaturing gradient gel electrophoresis (DGGE), and exploits the differential melting of mutant and wild-type PCR-amplified DNA fragments during electrophoresis through a temperature gradient. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the melting points (Tms) of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The temperature increment applied is typically minute (1 degree -1.5 degrees C) and the sweep speed is rather shallow (e.g., 0.05 degree C/min). Additionally, the denaturing thermal gradient is not controlled externally, but generated internally by Joule heat produced by voltage ramps. Point mutants are fully resolved into a spectrum of four bands, with a dynamic range extending from 45 degrees C (low melters) up to 70 degrees C for high melters. The present method can thus be universally applied to any type of point mutation.

DNA↗

PubMed2005

Inflammatory activity: capillary electrophoresis provides more information than erythrocyte sedimentation rate.

BACKGROUND: A new automated multicapillary zone electrophoresis instrument with improved resolution buffer (Capillarys with HR buffer, SEBIA, Paris, France) for analysis of human plasma proteins was compared with erythrocyte sedimentation rate (ESR). ESR determinations have been performed for more than eighty years and it is still one of the most frequently used laboratory tests, mainly to monitor the inflammatory response and as a tumour marker. METHODS: We studied the relationships between ESR, capillary electrophoresis and nephelometric determination of fibrinogen and albumin in 503 consecutive patient samples. The samples were analyzed on the Capillarys. The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis electropherogram. RESULTS: We found no significant correlation between presence or size of M-components and ESR. There were moderate to strong correlations between ESR, fibrinogen and capillary electrophoretic determination of alpha1-antitrypsin, alpha1-acid glycoprotein or haptoglobin for the detection of acute phase response. CONCLUSIONS: We suggest that ESR could be replaced by capillary electrophoresis for the assessment of inflammatory conditions and to detect M-components.

Acute-Phase Proteins↗

PubMed1976

Evaluation of heterogeneity in hemoglobins of Gallus domesticus and coturnix quail using disc-gel electrophoresis.

The heterogeneity in the hemoglobins of Gallus domesticus and Coturnix quail (Japonicum) was studied. Comparisons between a mutant hemoglobin type, the normal hemoglobin type and a heterozygous type were made in both species using disc-gel electrophoresis. The same heterogeneity of the primary hemoglobins as had previously been observed in these populations of chickens and quail by cellulose acetate electrophoresis was also observed using disc-gel electrophoresis. The same major hemoglobin component was observed in all the hemoglobin types, but the electrophoretic mibility of the minor component was different. In addition to the major and minor components which accounted for 93% of the total hemoglobin, four trace components were observed. One of these trace components (T) had previously been shown by cellulose acetate electrophoresis to be associated with the mutant minor hemoglobin. The other three trace components were in close proximity, but migrated anodally to the major, normal minor and mutant minor components. Their presence was not consistent and their concentration was usually at the lower limit of resolution. No differences could be detected between the electrophoretic mobility of the mutant hemoglobin of chickens and quails, suggesting that parallel mutations have occurred in the two species.

Animals↗

PubMed2000

Detection of the beta s gene: an evaluation of the solubility test against automated chromatography and haemoglobin electrophoresis.

The solubility test is evaluated against automated high-performance liquid chromatography (HPLC) and haemoglobin (Hb) electrophoresis for its efficacy in screening for the beta s gene in population groups in remote areas. Blood samples taken from 3246 individuals from the tribal populations of the Dhule and Gadchiroli districts of Maharashtra state were analysed by all three methods. The solubility test detected 871 out of 932 individuals positive for the beta s gene by HPLC and Hb electrophoresis, and showed an overall sensitivity of 93.8% and specificity of 100%, with a positive predictive value of 100% and negative predictive value of 97.4%. Both HPLC and Hb electrophoresis are relatively expensive and not available in most laboratories in remote tribal areas, where the frequency of the beta s gene is very high. We conclude that the solubility test could be used for preliminary screening to determine the prevalence of the beta s gene in different population groups, particularly in remote areas where other facilities are not available. Individuals who test positive for the beta s gene by the solubility test require further investigation by either HPLC or Hb electrophoresis.

Anemia, Sickle Cell↗

PubMedDate not supplied

[Therapeutic efficacy of electrophoresis of humic acids from sea mud in osteoarthritis].

Therapeutic action and efficiency of electrophoresis of humic acids (HA) from Khaapsaluskaya sea mud were studied in 97 patients with osteoarthrosis in comparison with electrophoresis of physiologic salt solution (PS). HA electrophoresis was found more effective (p < 0.01) as providing more potent anesthetic effects and more beneficial effect on joint function than PS electrophoresis (p < 0.005 and p < 0.01).

Case-Control Studies↗

PubMedDate not supplied

Monitoring the purity of a synthetic peptide by capillary electrophoresis: utilization of an on-line preconcentration method for improved separation and detection sensitivity.

To date, there has been a considerable amount of interest and success in the pharmaceutical industry in the discovery of drug targets and diagnostics utilizing peptides. The success of peptide pharmaceuticals has, however, been accompanied by some failures, both prior to entry and in the clinic. Progress has been made in various areas to improve the effectiveness of the final drug product. One major advance has been in the area of peptide synthesis and control of the purity of the peptide of interest. Recent advances in analytical instrumentation, including advances in capillary electrophoresis, have had a great impact on the ability to separate and detect low quantities of impurities and degradation products during the synthesis of a peptide drug. In this work, affinity capillary electrophoresis (ACE) was developed for the identification and characterization of a chemically synthesized peptide fragment of a snake toxin called fasciculin. The affinity capillary electrophoresis technology utilized in this study employed two powerful techniques coupled on-line for the direct and rapid determination of analytes in simple and complex matrices. The first technique, aimed for the nonselective extraction and concentration of one or more analytes of interest, utilizes a solid-phase analyte concentrator device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated target analyte(s), after elution from an analyte concentrator device. The on-line preconcentration step has proved valuable in terms of improving separation conditions as well as enhancing detection sensitivity values for the peptide fragment with a sensitivity increase ranging from 100- to 10,000-fold. Different types of analyte concentrator devices and a few binding-desorption conditions were tested. Bare and internal-wall-coated fused-silica capillaries were used. Comparative performance with HPLC in terms of selectivity and sensitivity is also discussed.

Chromatography, High Pressure Liquid↗

PubMed2001

[A case of IgA2-lambda type M-protein that IgA concentration differs from the values of M-protein by serum protein electrophoresis].

We report an IgA-lambda type M-protein in which the IgA concentration differed from the values of M-protein by serum protein electrophoresis found in a 53-year-old man with multiple myeloma. The M-protein value as determined by serum protein electrophoresis was 6,170 mg/dl. However, the serum IgA concentration was 3,052 mg/dl by turbidimetric immunoassay. Immuno-fixation electrophoresis using IgA subclass antisera revealed that this M-protein was the IgA2-lambda type. Western blotting analysis showed that the IgA2 molecules were composed of two approximately 68 kDa alpha 2 chains and two 28 kDa lambda chains. In addition the free lambda chain band was detected at the position of 28 kDa without 2-mercaptoethanol(2-ME) even though the patient IgA was purified. Since it is known that IgA2m(1) allotype easily release light chains from the IgA molecules in SDS-PAGE without 2-ME, we speculated that in this patient the IgA was the IgA2m(1) allotype. After peripheral blood stem cell transplantation(PBSCT), immunofixation electrophoresis of the patient serum revealed not only the bands of IgA2-lambda type M-protein, but also three bands of IgG1-kappa type M-protein in the gamma region.

Blood Protein Electrophoresis↗

PubMed1976

Resolution of erythrocyte membrane proteins by two-dimensional electrophoresis.

A two-dimensional electrophoresis method has been developed which solubilizes erythrocyte membrane proteins, and which resolves the components of the band that migrates in detergent gels as if its molecular mass were 95,000 daltons. This method uses gel electrophoresis with sodium dodecyl sulfate in the first dimension and phenol, aqueous urea, and acetic acid in the second dimension. The 95,000 dalton band is known to contain several different membrane proteins, including those associated with anion transport, glucose transport, and (Na+,K+) transport. Two-dimensional electrophoresis resolved this band into one major spot and several minor ones. Pronase digestion of whole erythrocytes, followed by preparation of ghosts and two-dimensional electrophoresis, showed that only the major component of this band was digested by pronase.

Blood Proteins↗

PubMed2002

[Study on the electrophoresis finger-prints of three groups of traditional Chinese drugs].

OBJECTIVE: To identify three groups of traditional Chinese drugs by electrophoresis finger-prints. METHODS: The 3 groups of drugs of different species or varieties were analyzed by protein, peroxidase(POD) isozyme and esterase (ES) isozyme polyacrylamide gel electrophoresis, respectively. And their finger-prints were established. RESULTS: Not less than one kind of electrophoresis finger-prints has significant difference between the drugs of each group, which can be used to distinguish all the species in this group. CONCLUSIONS: Different species even different varieties of traditional Chinese drugs can be identified accurately with proper electrophoresis finger-prints.

Drugs, Chinese Herbal↗

PubMedDate not supplied

High-throughput genotyping by microchip electrophoresis.

Easy applicability of modern microfabrication technology to electrophoresis microchips has initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field-mediated separations in microfabricated devices are significantly faster than conventional electrophoresis methods and are usually completed in seconds to minutes. The flexibility of fluidic manipulations in electrophoresis microchips allows the use of a variety of separation techniques and conditions. In this study, large-scale genotyping of the repeat polymorphism in the regulatory (promoter) region of the serotonin transporter gene 5-HTT linked polymorphic region (5-HTTLPR) was attempted using polymerase chain reaction (PCR) amplification followed by rapid microchip electrophoresis analysis of the amplicons.

DNA↗

PubMed2003

[Experimental research on human spermatozoa membrane proteins by two-dimensional gel electrophoresis].

OBJECTIVES: To analyze human spermatozoa membrane proteins by two-dimensional gel electrophoresis and to provide a basis for drawing the protein map of normal human spermatozoa membrane proteins. METHODS: Spermatozoa were purified by Percoll density centrifugation, and spermatozoa membrane proteins were analyzed by two-dimensional gel electrophoresis using isoelectric focusing and polyacrylamide gel electrophoresis. RESULTS: About 800 protein spots could be identified by the imaging analysis system. The molecular weight and isoelectric point of most proteins were 20,000 to 100,000 and 3.0 to 7.0 respectively. CONCLUSIONS: There are more than 800 types of proteins in the human spermatozoa membrane. The spermatozoa membrane protein can be identified with a certain precision by two-dimensional gel electrophoresis.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1992

Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.

Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow.

Electrophoresis, Polyacrylamide Gel↗