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PubMed1989

Electrophoresis of DNA in oriented agarose gels.

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.

DNA↗

PubMed1988

BBC microcomputer controlled field inversion gel electrophoresis.

Agarose gel electrophoresis to separate DNA molecules is a widely used technique in molecular biology but there is an upper limit to the sizes that can be resolved. Pulsed field techniques have extended this limit but require expensive equipment. Here we describe a home-made control unit to interface conventional electrophoresis equipment to a BBC microcomputer for the purposes of field inversion gel electrophoresis.

Electrophoresis↗

PubMed2000

DNA electrophoresis in a monodisperse porous medium.

Electrophoresis of DNA migrating in an ordered matrix is studied and compared with classical agarose gel electrophoresis. A well-defined migration medium is obtained by crystallization of monodisperse silica spheres. Electrophoretic mobility of DNA is measured with fluorescence recovery after photobleaching experiments. The main result is that, as it was the case for gel electrophoresis, diffusion and electrophoretic mobility of DNA in such a medium are well described with reptation theories.

Base Pairing↗

PubMed1985

Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation.

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

Animals↗

PubMed1972

Application of an improved system of electrophoresis in acrylamide gel to studies on the sera of different species.

Polyacrylamide gels constitute a generally better matrix for routine electrophoresis of serum proteins than other media commonly employed, but the immunoglobulin fractions of the largest size may not migrate into gels in which the acrylamide concentration exceeds 5%. To facilitate adequate separation, slabs containing a semi-solid layer were prepared from discontinuous gels consisting of 2 to 8% acrylamide. Serum samples were subjected to electrophoresis, under carefully controlled conditions at pH 9.0, by means of a pulsed constant power supply. The method provided a rapid, reproducible, and relatively simple technique for the study of human serum proteins, either by electrophoresis or by immunoelectrophoresis, and for differentiation of serum samples from various animal species.

Animals↗

PubMed1976

Comparative zone electrophoresis of catalase of Staphylococcus species isolated from mammalian skin.

Vertical polyacrylamide slab gel electrophoresis was conducted for the catalase enzymes of representative strains of 18 proposed species and subspecies of the genus Staphylococcus. The catalase bands which resulted were predominantly monomorphic within each of the species and differences in catalase mobilities were observed between many of the species. The electrophoretic mobilities of the catalases were supportive to the scheme of classification used. Many strains of certain species demonstrated multiple catalase bands which are suggestive of multimolecular forms of the enzyme. Horizontal starch gel electrophoresis of representative strains of S. capitis produced catalase bands with relative mobilities that were different from those obtained with polyacrylamide electrophoresis, presumably due to a difference in molecular sieving between the gels.

Animals↗

PubMed2004

Detection of c-kit mutations in canine mast cell tumors using fluorescent polyacrylamide gel electrophoresis.

Mutations consisting of internal tandem duplications (ITDs) in exons 11 and 12 of the proto-oncogene c-kit are found in 30-50% of malignant canine mast cell tumors (MCTs). Traditionally, identification of such mutations in tumor specimens has been undertaken using standard polymerase chain reaction (PCR) and agarose gel electrophoresis. This procedure is limited to the detection of insertions and deletions larger than 9 base pairs in size. The purpose of this study was to compare the efficiency and accuracy of standard agarose gel electrophoresis with fluorescent polyacrylamide gel electrophoresis (PAGE) for the detection of ITDs in canine MCTs. The results of this study demonstrate that PAGE of labeled PCR products accurately predicts the size of the ITD in each tumor. In addition, other small insertions and deletions were not identified, suggesting that if they occur in canine MCTs, they do so infrequently. Because fluorescent and polyacrylamide formats are automated and have better resolution than agarose gels, fluorescent PAGE provides a more accurate, economical, and higher throughput method for the detection of c-kit mutations in canine MCTs.

Animals↗

PubMed1985

An application of the high-iron diamine staining for detection of sulfated glycoproteins (glycopeptides) in electrophoresis on cellulose acetate membrane.

The high-iron diamine staining (HID), which has been used in histochemistry to stain sulfated glycoconjugates (SGC), was tested for detectability of authentic acidic substances (chondroitin sulfates A plus C, dermatan sulfate, heparan sulfate, chondroitin, hyaluronic acid, alpha 1-acid glycoprotein and ribonucleic acid) in electrophoresis on cellulose acetate membrane (Separax). The results showed that only SGC were detectable by the HID, although all the above substances were stained with alcian blue. The glycoconjugate preparations obtained from the liver, kidney, lung, small intestine, colon, stomach, brain and spleen of rats were examined by two-dimensional electrophoresis on Separax. The new spots (or bands), besides those of sulfated glycosaminoglycans, were detected by the HID on the electrophoretograms of all the samples except for the kidney one. The substances giving the new spots (or bands) were indicated to be sulfated glycopeptides (SGP) by crude heparinase digestion of a representative sample. The present results revealed that the HID was applicable for detection of SGP in electrophoresis on cellulose acetate membrane. Also, it is a novel finding that the liver and spleen contain SGP.

Animals↗

PubMed2001

Electrophoresis gel image processing and analysis using the KODAK 1D software.

The present article reports on the performance of the KODAK 1D Image Analysis Software for the acquisition of information from electrophoresis experiments and highlights the utility of several mathematical functions for subsequent image processing, analysis, and presentation. Digital images of Coomassie-stained polyacrylamide protein gels containing molecular weight standards and ethidium bromide stained agarose gels containing DNA mass standards are acquired using the KODAK Electrophoresis Documentation and Analysis System 290 (EDAS 290). The KODAK 1D software is used to optimize lane and band identification using features such as isomolecular weight lines. Mathematical functions for mass standard representation are presented, and two methods for estimation of unknown band mass are compared. Given the progressive transition of electrophoresis data acquisition and daily reporting in peer-reviewed journals to digital formats ranging from 8-bit systems such as EDAS 290 to more expensive 16-bit systems, the utility of algorithms such as Gaussian modeling, which can correct geometric aberrations such as clipping due to signal saturation common at lower bit depth levels, is discussed. Finally, image-processing tools that can facilitate image preparation for presentation are demonstrated.

Algorithms↗

PubMed1989

Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen.

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Animals↗

PubMed1992

Tear protein G originates from denatured tear specific prealbumin as revealed by two-dimensional electrophoresis.

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS PAGE) of the non-denatured low molecular weight (MW) tear proteins (dilution in phosphate buffered saline or in the non-ionic detergent Triton X100) revealed no protein G but a strongly marked 23-kD related to a tear specific prealbumin (TSP) subunit coming with the known 15, 17, 18 and 20-kD TSP subunits. Under mild denaturating conditions of sample preparation with SDS dilution just before electrophoresis, 23-kD protein decreased and a faint 32-kD protein G appeared. Under stronger denaturing conditions of sample preparation with SDS treatment (boiling or freeze-thaw cycles), 23-kD protein disappeared and two main protein G forms (32 and 34-kD) and additional bands (29, 36, 39, 42, 57 and 60-kD) appeared depending on the sample treatment. The isoelectric pH (pI) of these proteins ranged from pH 5.2 to pH 5.4. Different two-dimensional electrophoresis methods revealed that: - in presence of SDS, 23-kD protein was spontaneously changed into 17-kD TSP and such a phenomenon was partially reversible by using a non-ionic detergent (Triton X100), new proteins appeared under denaturating processes were related to various protein G forms and originated from TSP group, proteins G were produced by the aggregation of TSP subunit related to MW 17-kD/pI 5.0 corresponding to the major TSP subunit, disulfide bond formation was shown to play a major role in the aggregation process although protein G group was not totally reduced by dithiothreitol. Such results suggest that protein G is an in vitro experimental artifact due to denaturing conditions with SDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1977

Structure in relation to behavior of mutant hemoglobins in citrate agar electrophoresis.

The comparative mobilities, in citrate agar electrophoresis, of 91 mutant hemoglobins are presented in relation to their molecular structure and in some cases, to their mobilities in other types of electrophoresis. More than a third of the alpha chain mutants (11 of the 27 examined) and half of the beta chain mutants (29 of 55) differ to some extent from Hb A. The helical location of the substituted residue is an important determinant of hemoglobin mobility, which is also affected by a complex interplay of other factors. When the data are combined with those of several other types of electrophoresis, they often provide presumptive (or in some cases highly specific) identifications of mutant hemoglobins and hemoglobinopathies.

Amino Acids↗

PubMedDate not supplied

A preliminary evaluation of serum proteins by capillary electrophoresis.

We evaluated the 5-band Serum Proteins by Capillary Electrophoresis kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) on the BioFocus 2000 CE (Bio-Rad) against conventional agarose gel electrophoresis (AGE) (Helena Laboratories, Beaumont, TX, U.S.A.). Serum from 60 patients was initially screened by AGE and divided into three groups: 1) normal electrophoretic pattern (n = 36, mean total protein 67.7 g/L), 2) monoclonal/oligoclonal gammopathy (n = 14, mean total protein 78.8 g/L), and 3) polyclonal gammopathy (n = 10, mean total protein 77.4 g/L). These samples were concurrently analyzed on the BioFocus 2000. Intraassay and interassay CVs for the five fractions for a normal sample were 0.17-1.44% and 0.42-9.11%, respectively, and 0.21-3.37% and 0.29-3.61%, respectively, for a sample with monoclonal gammopathy. Correlation coefficients for albumin and the albumin/globulin (A/G) ratio were 0.8891 (albumin range 17-93 g/L) and 0.8276 (A/G ratio range 0.39-7.81), respectively. The A/G ratio alone could not discriminate between the three groups. Capillary zone electrophoresis (CZE) correctly identified 33 of 36 samples in the normal group, and 22 of 24 samples in the other two groups, giving a clinical sensitivity and specificity of 91.7%. Our preliminary evaluation shows that protein separation by CZE is a simple, rapid, and automated alternative to conventional AGE.

Adult↗

PubMed1999

[Determination of serum proteins by high performance capillary zone electrophoresis].

The separation method of serum proteins was established with an untreared 50 microns i.d. x 47 cm (40 cm to detector) capillary and detection of absorbance at 200 nm. Analysis was performed by pressure injectction 17.23 kPa.s and by applying 23 kV in the constant voltage mode. Serum samples were diluted 40-folds with assay buffer (12.5 mmol/L sodium borate, 1 mmol/L calcium lactate, 0.7 mmol/L magnesium sulfate, 1 mmol/L EDTA were mixed). A normal control serum protein was separated into 6 fractions. In pregnant serum, the alpha 0 was an additionally unknown fraction. Comparison of capillary electrophoresis with conventional cellulose acetate electrophoresis for analysis of serum proteins from normal control, pregnant women multiple myeloma and tonic rachitis patients indicates that capillary clectrophoresis is a new technique for the analysis of serum proteins because of its high efficiency, on-line data processing and automation. Capillary electrophoresis is the reliable technique for clinical diagnosis of serum protein abnormalities.

Albumins↗

PubMed1992

[High resolution electrophoresis of serum aand amniotic fluid].

In a sample group of 92 women undergoing prenatal echo-guided transabdominal amniocentesis between the 12th and 23rd week of pregnancy the Authors analysed amniotic fluid and maternal serum using the recently developed method of high resolution protein electrophoresis in order to identify the presence of particular proteins in the amniotic fluid which are pathognomonic for a number of maternofetal pathologies. The results obtained in normal and pathological pregnancies or in the case of twins showed a marked dispersion in amniotic fluid of total protein concentrations depending on the period of gestation; in addition, albumin, alpha 1-antitrypsin, alpha 2-glycoprotein acid, alpha 2-macroglobulin and beta 2-protein were also found. Plasma levels of prealbumin, albumin, alpha 1-glycoprotein acid and IgG were slightly reduced, whereas there was a marked increase in ceruloplasmin, transferrin and fibrinogen; C3 and haptoglobin levels were normal. It is therefore possible to ascertain that amniotic fluid proteins analysed by high-resolution 15-band electrophoresis did not vary qualitatively or quantitatively until the 23rd week of gestation and in those cases of twin or pathological pregnancies examined no anomalous band was found in the protein electrophoresis of maternal serum or amniotic fluid which might prove useful in prenatal diagnosis.

Amniotic Fluid↗

PubMed1992

Two-dimensional electrophoresis of urine specimens from patients with renal disease.

In an effort to develop non-invasive markers capable of characterizing renal disease with greater sensitivity and specificity than those currently available, urinary proteins were studied by high-resolution two-dimensional electrophoresis in order to catalog those proteins which appeared to be most affected by a variety of renal diseases. Urine specimens were prepared by high-pressure liquid chromatography and subjected to two-dimensional gel electrophoresis. Proteins were visualized with silver stain. Gels from 17 patients were analyzed in detail and compared to standard maps of human urinary proteins and plasma. The two-dimensional gels displayed approximately 100 times the number of proteins demonstrated by high resolution agarose electrophoresis. We have identified 34 proteins whose urinary concentrations were most affected by renal disease. Twenty seven were of plasma origin, based upon comigration with plasma samples (17 of unknown identity and function). Seven proteins were observed in normal urine but not present in plasma. These proteins, which may represent kidney tissue proteins, were not apparent in the urine of patients with proteinuria, most likely due to dilutional effects.

Biomarkers↗

PubMed2005

[Capacitively coupled contactless conductivity detection in capillary electrophoresis].

Capacitively coupled contactless conductivity detection (C4D) is a new detection technique has been developed in recent years and used mainly in capillary electrophoresis and microchip electrophoresis. The characteristics of C4D detector are simple in structure, easy in miniaturization and integration, and free of electrodes contamination, which are common problems in an electrochemical detection. The principles and progresses of C4D in capillary electrophoresis are reviewed.

Conductometry↗

PubMed1985

[2-dimensional electrophoresis of cerebrospinal fluid in various neurological patients].

At present two-dimensional (2D) electrophoresis is the best method of resolution for separating complex protein mixtures, and this method has been successfully adapted for resolving CSF proteins. CSF from over 200 patients with a wide spectrum of neurological diseases were analyzed by 2D electrophoresis in addition to agarose electrophoresis. After a 2D run several hundred fractions of proteins were obtained. By perfecting this method the authors have succeeded in demonstrating oligoclonal zone patterns not previously described in the light chain region of the immunoglobulins in CSF samples. All patients with definite diagnosis of multiple sclerosis (MS), and also other diseases such as neurolues, myelitis, herpes zoster, etc. showed particular oligoclonal zone patterns. This experience indicates that the detection of oligoclonal zones in the region of the Ig-light chain is of major diagnostic significance. Moreover, the region of the so-called "CSF-specific" proteins deserves special attention.

Adult↗