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PubMedDate not supplied

Evaluation of proteinuria by two-dimensional polyacrylamide gel electrophoresis after kidney transplantation.

Urinary proteins were studied in patients after kidney transplantation during various functional states (normal function, acute rejection, chronic rejection) by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, the proteins were separated according to their electric charge by isoelectric focusing, and in the second dimension according to their molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis the proteins were visualized using a highly sensitive silver stain technique. The combination of these methods allowed the discrimination of up to 250 protein spots in human urine, most of them unidentified up to now. Functional changes of the kidney transplants were accompanied by complex changes of the protein pattern in urine. These changes cannot be simply compared to the tubular and glomerular pattern of proteinuria which can be identified by one-dimensional sodium dodecyl sulfate electrophoresis. The 2D electrophoresis of urinary proteins may develop into a useful tool in localizing of kidney damage and in the evaluation of renal disease.

Albuminuria↗

PubMed2004

[Establishment of two-dimensional electrophoresis of uterine leiomyoma for the proteome analysis].

OBJECTIVE: To establish and optimize the two-dimensional electrophoresis (2-DE) of uterine leiomyoma for the proteome analysis. METHODS: Run immobilized pH gradient (IPG)-isoelectric focusing electrophoresis as the first dimension, then vertical SDS-PAGE electrophoresis as the second dimension. A series of important steps,such as sample solubility, volume of loading, electrophoresis parameters and protocol for staining were optimized. RESULTS: The 2-DE patterns of uterine leiomyoma and myometrium with good quality were obtained. CONCLUSION: With optimal condition the two-dimensional electrophoresis of uterine leiomyoma can be obtained.

Electrophoresis, Gel, Two-Dimensional↗

PubMedDate not supplied

Chemical degradation of tobacco mosaic virus followed by infectivity assay, reverse transcription-polymerase chain reaction and gel electrophoresis.

In order to determine the detection limit for chemically treated virions by gel electrophoresis, reverse transcription-polymerase chain reaction (RT-PCR) and infectivity assay, tobacco mosaic virus (TMV) exposed to various concentrations of chemicals was studied. When virions were exposed to 0.2 N HCl for 30 mins, partially degraded TMV particles were observed by gel electrophoresis. Under the same exposure, a major RT-PCR amplified DNA product corresponding to the target size of 806 bp, which decreased as a function of time, could be detected for up to 60 mins of exposure. When virions were treated with NaOH (0.02 N or higher normality) for 5 mins, partially degraded virions were detected by gel electrophoresis, exhibiting multiple band patterns. Exposure of the virions to 0.1 N NaOH for 5 mins revealed severely degraded viral RNA, but disappearance of the amplified RT-PCR products was apparent during 30-60 mins of exposure. Therefore, these data showed clearly the difference in the detection limit of gel electrophoresis and that of RT-PCR for the degraded viral RNA. In addition, the infectivity assay showed that the number of local lesions in Nicotiana rustica were significantly reduced by more than 95% when the virus was exposed to 0.2 N HCl for 15 mins or 0.1 N NaOH for 10 mins. From these results we conclude that loss of infectivity was not related to that of PCR product. Other chemical disinfectants such as phenol or formalin were also found to be effective to reduce the virus infectivity, but a corresponding degradation of viral RNA was detected by neither gel electrophoresis nor RT-PCR.

Electrophoresis, Agar Gel↗

PubMed2002

[Electrophoresis and the traditional method for the measurement of lipoprotein cholesterol in serum].

OBJECTIVE: To compare the advantages and disadvantages of electrophoresis with the traditional method in measuring lipoprotein cholesterol in serum. METHODS: Four lipoprotein cholesterols were determined by the Helena electrophoretic system. Quantification of high density lipoprotein-choleterol(HDL-C) was performed by phosphotungstate-magnesium precipitation or direct HDL-C assay. The concentration of low density lipoprotein-cholesterol (LDL-C) was calculated by F-equation. RESULTS: Four lipoprotein zones were segregated distinctly by the electrophoretic method. Lp(a) zone appeared only in 6 out of 68 normal samples (8.82%). Reference values of HDL-C, Lp(a)-C, VLDL-C, and LDL-C in serum were 1.16-2.19, 0-0.22, 0.07-0.47, and 1.86-3.46 mmol.L-1, respectively. When comparing the results by electrophoresis with those by the precipitation method for HDL-C, no significant difference was found (P > 0.05); but there was a significant difference between electrophoresis and F-formula calculation for LDL-C (P < 0.05). When comparing results of electrophoresis with the direct method, we found significant differences in both HDL-C and LDL-C (P < 0.01). CONCLUSION: F-formula used for calculating LDL-C is not appropriate to clinical application. Electrophoresis allows the quantification of HDL-C, VLDL-C, LP(a)-C, and LDL-C. The method is simple, convenient, and appropriate to routine application.

Adult↗

PubMed2006

Pharmaceuticals analysis by capillary electrophoresis at nanolevel detection.

There is an urgent need to analyze drugs and pharmaceuticals at nanoscale detection, both now and in the future. Capillary electrophoresis is a growing technique that is continuously developing with improved detectors, sample preparation units, and other components. This article describes the analysis of drugs and pharmaceuticals by capillary electrophoresis at nanoscale detection in various standard solutions, dosage formulations, and biological samples. Nanoscale detection analysis is discussed in terms of the advancement in the detectors and sample preparation methods. The coupling of the detectors and sample preparation units to capillary electrophoresis is also discussed. Attempts have been made to evaluate the optimization strategies of capillary electrophoresis conditions to achieve detection at the nanoscale. Applications of capillary electrophoresis for the analyses of simple and chiral mixtures of several drugs and pharmaceuticals at nanoscale detection are included.

Electrophoresis, Capillary↗

PubMed1991

Separation and detection of DNA polynucleotides using capillary electrophoresis. Application to detection of polymerase chain reaction-amplified human immunodeficiency virus and HLA DNA.

The polymerase chain reaction (PCR) is a technique for rapid amplification of target DNA sequences. During the past several years, a large number of research applications of PCR have appeared, many of which may prove to be useful clinically. We report the use of capillary electrophoresis, a fully automated technique, as an alternative to polyacrylamide gel electrophoresis for the detection of PCR-amplified viral and cellular DNA. We describe conditions for rapid separation, detection, and discrimination of PCR products from the human immunodeficiency virus type 1 gag gene and the HLA-DQ-alpha gene amplified from the human immunodeficiency virus provirus-containing U1.1 cell line. The sensitivity achieved with the use of capillary electrophoresis analysis was roughly equivalent to that of ethidium bromide staining of polyacrylamide gel electrophoresis gels. Further refinement of capillary electrophoresis for automated detection and quantitation of PCR-amplified products should expedite more widespread application of PCR analysis in the clinical laboratory.

DNA↗

PubMed1991

[Capillary electrophoresis: principle and applications].

Capillary Zone Electrophoresis is a new separation technique which is performed in capillaries (ID smaller than 100 microns). The separations are carried out according to several modes which are mainly the Free Solution Capillary Electrophoresis, the Micellar Electrokinetic Capillary Chromatography and the Gel Capillary Electrophoresis. Many detection systems can be used. This technique has many advantages: high separative power, short time of analysis, low cost of operation and very low sample consumption. Capillary electrophoresis and HPLC appear to be complementary because they are based on different physical and chemical properties. Capillary electrophoresis has a great potential of applications for both chemical and biochemical analysis. This new and very efficient analytical technique is of growing interest for biochemical analysis and its use is likely to increase rapidly.

Biological Assay↗

PubMed1988

Extraction of proteins within ultrathin-layer polyacrylamide electrophoresis (SDS-PAGE) and isoelectric focusing (PAGIF) of cryostat sections and tissue culture specimens.

Separations on a micro- and ultramicroscale by electrophoresis and isoelectric focusing of protein quantities between 10(-6) and 10(-12) g have notable interest, particularly in cytology and histology. We performed the methods of ultrathin-layer polyacrylamide horizontal electrophoresis, isoelectric focusing and protein mapping with some modifications in order to study protein extraction and applied them for cryostat sectioned muscle tissues and singular dorsal root ganglia of the chicken in tissue culture. Further we established an extraction chamber for histological specimens. The basis for ultrathin-layer electrophoresis is the ultrathin (0.12 mm-0.36 mm) polyacrylamide gel on glass plates or microscopic slides. This method allows a considerable reduction of the amount of proteins at the range of 10(-9) g and is thus appropriate for direct extraction of proteins within electrophoresis (10 min, 5 mA 200 V) or isoelectric focusing (10 min, 10 mA, 500 V) of cryostat-sections (7-20 micron thickness) or tissue culture specimens. The advantages of these techniques for extraction of soluble proteins in immunohistochemistry as well as for handling to obtain optimal resolution (compared with electrophoresis of conventional extracted proteins) will be demonstrated.

Animals↗

PubMed1985

Immunoenzymetric assay for creatine kinase MB with subunit-specific monoclonal antibodies compared with an immunochemical method and electrophoresis.

Results of an immunoenzymetric assay (TANDEM-E CKMB) for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme, in which subunit-specific monoclonal antibodies are used, were compared with those by an immunochemical method (Isomune-CK) and electrophoresis (Corning agarose gel). The study involved 200 patients; greater than 500 samples were analyzed by all three methods. The analytical performances were acceptable. Between-method correlation coefficients ranged from 0.881 to 0.975. Two reference intervals were established for the immunoassays: 0-4 micrograms/L (TANDEM) and 0-4 U/L (Isomune) for "normal" patients; 0-9 micrograms/L (TANDEM) and 0-14 U/L (Isomune) for noninfarct patients. Agreement with respect to increased CK-MB as defined by the reference intervals for the noninfarct patient was 96% between TANDEM and electrophoresis, 90% between Isomune and electrophoresis. All three methods are acceptable for use in determining CK-MB, but the overall diagnostic efficiencies for the mass or activity concentration of the isoenzyme and for its proportion of total CK activity, based on the predictive value model, are 92% (electrophoresis, 0-7 U/L), 90% (electrophoresis, 0-4%), 92% (TANDEM, 0-9 micrograms/L), 88% (TANDEM, 0-3% index), 88% (Isomune, 0-14 U/L), and 83% (Isomune, 0-4%). All three methods can detect CK-MB in serum, but its presence is not necessarily diagnostic of acute infarct. We recommend using the actual concentration of CK-MB to evaluate patients with suspected acute myocardial infarct, and the percentage of CK-MB when total CK is very high.

Antibodies, Monoclonal↗

PubMed1980

Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Enzyme activity was detected in situ by action on substrates introduced into the gel and subsequent staining of either the product or unreacted substrate. This technique combines the advantage of enzyme identification with the resolution and molecular weight dependence of gel electrophoresis in the presence of sodium dodecyl sulfate. Enzymes appeared to recover activity as soon as the detergent diffused out of the gel. Most monomeric enzymes could be renatured even after disruption of their disulfide bonds, but several proteases, including trypsin, could not. Oligomeric enzymes composed of identical subunits were poorly renaturable. Renatured enzymes were retained in gels after electrophoresis longer than native enzymes which had been subjected to electrophoresis in the absence of detergent. Re-electrophoresis of the renatured enzymes showed that part of the retained activity was physically anchored to the gel, possibly by the folding of polypeptide aroung the gel matrix as the enzymes were renatured.

Amylases↗

PubMed1982

[Electrokinetic property of brain tumor cells by the aid of free flow electrophoresis (author's transl)].

Malignant transformation in tumors is characterized by changes in cell surface components such as glycolipids and glycoproteins and is determined by the sialic acid content of the glycoconjugates. Our purpose was to investigate whether or not a similar change occurred in the brain tumor cells during malignant transformation. Change in the electrical charge of the cell surface was studied by means of cell electrophoresis. Experimental cell lines of rat gliomas and fibroblasts were cultured and compared for electrical charge by cell electrophoresis. Four various grade gliomas and three meningiomas from human subjects were cultured and compared for electrical charge by cell electrophoresis. Free flow electrophoresis (Elphar Vap 5) was used because it characteristically does not interfere with the use of the cells for biochemical and biological studies following electrophoresis and because it also allows the possibility of subsequent cell culture. The rat glioma cells had lower electrical charges than the fibroblasts, and the human malignant brain tumor cells exhibited lower electrical charges than the benign tumor cells. These findings suggest a lower sialic acid content in malignant than in benign tumors and agree with theories which postulate a decrease in sialic acid content accompanying malignant transformation. This differentiation between benign and malignant tumors was made on the basis of the cell surface electrical charge and could be used for clinical diagnosis and treatment.

Animals↗

PubMed1994

High-performance capillary electrophoresis of human serum and plasma proteins.

In recent years, capillary electrophoresis has proved extremely useful for separation of biomolecules such as nucleotides, amino acids, peptides, and proteins. The advanced instrumental design of commercial CE equipment has offered several advantages to conventional electrophoresis. Precise sample application, temperature regulation, and stable, reproducible instrument control with respect to voltage, current, pressure, and run-to-run reproducibility is routinely achieved in contemporary commercially available equipment. Based on these instrumental features, CE is already used as a routine method for the analysis of small molecules. In contrast, serum and plasma proteins represent a difficult challenge for capillary electrophoresis. Routine analysis of serum and plasma proteins is reduced to the application of capillary zone electrophoresis, which proved to be a reliable method for rapid screening of whole serum, purity control of serum proteins and fractions, as well as on-line quantification of the components. With regard to the simplicity of the method and the low cost of operation, CZE proved to be an attractive alternative to comparable conventional methods. Other separation techniques, such as CITP, CIEF, and CGE, provide higher resolution of the sample proteins than that provided by CZE. Furthermore, information about physicochemical properties of the analyte, such as isoelectric point or molecular weight, can be gained by CIEF or CGE. Even though this information is essential for protein analysis and characterization, these methods are not commonly used at present because reliable methods for controlling the EOF are not commercially available. Coated capillaries are expensive, and it is still difficult to purchase a set of coated capillaries with identical properties. As long as the reproducibility from capillary to capillary cannot be guaranteed, the high resolving methods will not be applied in routine analysis. The development of reliable regulation of the EOF, either by an appropriate column material or coating or an external field control, will certainly lead to a breakthrough for the capillary electrophoresis of human serum and plasma proteins.

Blood Proteins↗

PubMed1998

Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser-induced fluorescence detection.

The optimum separation conditions of polymerase chain reaction (PCR) products have been found for high-speed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. DNA fragments obtained after PCR amplification of the region covering the (CA)18 microsatellite repeat in nitron 5 of the gene for FcERIbeta, a high affinity glycoprotein receptor for IgE, located on chromosome 11 (11q13), were analyzed with the aim of investigating the repeat polymorphism. The results of polyacrylamide slab gel electrophoresis (PAGE), agarose gel electrophoresis, CE with absorbance detector and CE with LIF are compared. The CE with LIF proved to shorten analysis time by a factor of 100 when compared to slab gel electrophoresis. CE-LIF utilizes a short capillary with an effective length of 6.3 cm and electric field strength from 100 to 550 V/cm. The respective PCR products of sizes from 116 to 210 base pairs (bp) were analyzed in 3 min.

Base Sequence↗

PubMedDate not supplied

Abnormal protein patterns in blood serum and cerebrospinal fluid detected by capillary electrophoresis.

Capillary zone electrophoresis and high-resolution agarose gel electrophoresis were compared to detect protein components in serum and cerebrospinal fluid of patients. Both electrophoretic methods proved to be useful for detection of protein abnormalities (e.g., mono- and oligoclonal bands) in biological fluids, but capillary electrophoresis offered several important advantages, such as sample application without preliminary concentration, lack of staining procedures, and on-line evaluation of patterns. Furthermore, capillary electrophoresis exhibits shorter analysis time and high resolution with low baseline noise. The results were proven to be powerful in diagnosis and monitoring of dyscrasias in routine laboratory practice.

Adolescent↗

PubMed1987

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage.

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.

Animals↗

PubMed1990

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.

Base Sequence↗

PubMed1980

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients.

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.

Animals↗

PubMed1998

Electrophoresis of long DNA molecules in linear polyacrylamide solutions.

Electrophoresis of long DNA (T4 DNA; 166 kb, S. pombe chromosomal DNA; 3-6 Mb) in linear polyacrylamide solutions was investigated by fluorescence microscopy and capillary electrophoresis. In the past studies on electrophoresis of long DNA in a polymer solution, it was reported that DNA migrates in 'U-shape conformation'. We found that at higher polymer concentrations, the shape of the migrating DNA changes from U shape to linear shape ('I-shape conformation'). In the migration mode with the I-shape conformation, the DNA moves with almost constant velocity and constant shape. However, the migration velocity does depend on the DNA size, and it is possible to separate DNAs under this I-shape motion. Actually, Mb-sized DNAs are well separated within 5 min in the region for the I-shape motion by means of capillary electrophoresis with a DC field. Considering that it takes 20 h to separate Mb-sized DNAs by standard pulsed-field gel electrophoresis (PFGE), this results will be useful for the separation of giant DNAs.

Journal Article↗