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Mutation detection using nucleotide analogs that alter electrophoretic mobility.

Kornher, J S; Livak, K J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/10/1989 EN
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A simple primer extension assay has been developed to distinguish homologous DNA segments differing by as little as a single nucleotide. DNA strands are synthesized with one of the four natural nucleotides replaced with an analog that affects electrophoretic mobility. DNAs that are the same length but differ in the number of analog molecules per strand exhibit different mobilities on a sequencing gel. In combination with the polymerase chain reaction (PCR; 1, 2), this method has been used to distinguish mutant and normal alleles of the human insulin receptor gene that differ by a single-base substitution. The method appears to be generally applicable to the detection of any nucleotide polymorphism in any segment of DNA.

An assessment of the macrophage electrophoretic mobility test (MEM) in cancer diagnosis.

Crozier, E H; Hollinger, M E; Woodend, B E; Robertson, J H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1976 EN
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The results of an assessment of the macrophage electrophoretic mobility test in cancer diagnosis are described. We found that the test did not reliably distinguish between a group of 18 patients with benign disorders and a group of 25 with malignant disease.

Evaluation of macrophage electrophoretic mobility (MEM) test as an indicator of cellular immunity in ocular tumours.

Rahi, A. H.; Otiko, G.; Winder, A. F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1976 EN
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The macrophage electrophoretic mobility (MEM) test of Field and Caspary did not clearly separate patients with ocular neoplastic disease from those with inflammatory disease, although there was some indication of discrimination between choroidal melanoma and ocular inflammation. In our hands the test failed to give a reproducible result for the immunodiagnosis of ocular malignancy. The technique, however, seems to provide some indication of delayed hypersensitivity in experimental inflammatory eye diseases when relatively pure antigens are used.

Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and β-Conglycinin Subunits 1

Fontes, Elizabeth Pacheco Batista; Moreira, Maurilio Aves; Davies, Corinne S.; Nielsen, Niels C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1984 EN
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Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A3 and A4 could be resolved from the other acidic polypeptides. A variant of the δ′ subunit of β-conglycinin was identified.

The electrophoretic mobility of peptides on paper at pH1.9 (Short Communication)

Bailey, C. J.; Ramshaw, J. A. M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1973 EN
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The relationship between the electrophoretic mobility, the molecular weight and the charge of peptides at pH1.9 was re-investigated. It was shown that the relationship at pH1.9 is quantitatively similar to that which holds at pH6.5.

Patterns of Molecular Variation. II. Associations of Electrophoretic Mobility and Larval Substrate within Species of the DROSOPHILA MULLERI Complex

Richardson, R. H.; Smouse, Peter E.; Richardson, Martha E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1977 EN
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Electromorphic variation among populations of Drosophila mojavensis, D. arizonensis and D. longicornis was examined for seven genetic loci. The average electrophoretic mobility for a population was used as the metric. D. mojavensis and D. arizonensis use larval substrates in different parts of their geographic ranges, while D. longicornis is more narrowly restricted to different species of the cactus Opuntia in different localities. There is marked electromorphic variation among populations of either D. mojavensis or D. arizonensis, and the bulk of this variation is accounted for by differences in laval substrate. There is somewhat less variation among populations of D. longicornis, and only a moderate portion of this is accounted for by larval substrate differences. There appears to be an association between the taxonomic diversity of the larval substrates and the electromorphic diversity of the Drosophila populations utilizing those substrates. Evidence is reviewed that suggests physiological mechanisms for these possibly adaptive associations.

Electrophoretic mobility and surface immunoglobin of albumin gradient fractionated mouse spleen cells.

Dumont F, 54500, France
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1975 EN
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Spleen cells from normal CBA mice, containing B and T lymphocytes, cyclophosphamide-treated CBA mice, containing almost exclusively T lymphocytes, and athymic nudemice, containing only B lymphocytes, were fracitonated by differential flotation in adiscontinuous albumin gradient. In all three cases, four density fractions were regularly obtained. The electrophoretic mobility (EPM), which allows distinction betweenslow-EPM (B) cells and fast-EPM (T) cells and the presence of surface immunoglobulins (sIg) detectable by direct immunofluorescence, characterizing B lymphocytes, were investigated on these fractions. Both B and T cells were recovered throughout the gradient, but in different proportions. Thus, B cells (slow-EPM, sIg-bearing) were enriched in the light density fractions while T cells (fast-EPM)were more numerous in the denser fraction. The mean EPM of slow-moving cells decreased, whereas that of fast-moving cells increased, as their buoyant density increased. Less nude spleen (B) cells were found to bear sIg in the light density fractions than in the denser fractions. These findings suggest the existence of lymphocyte subpopulations with distinct physicochemical properties which might represent stages in the maturation and differentiation of B- and T-cell lineages.

The mechanism of antigen-induced electrophoretic mobility reduction of guinea-pig macrophages

Caspary, E. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1972 EN
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The interaction of antigen with sensitized lymphocytes produces a protein substance capable of reducing the electrophoretic mobility of guinea-pig peritoneal macrophages. Reaction with the protein slowing factor also involves some vital synthetic activities of the macrophage. A critical number of lymphocytes is required to initiate the complete reaction suggesting that above this threshold recruitment may take place. It is suggested that the mechanism of action is a close parallel to that of macrophage migration inhibition.

Antigen cross-reactivity in the macrophage electrophoretic mobility test. A study using cellular affinity chromatography

McDermott, J. R.; Caspary, E. A.; Dickinson, J. P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1974 EN
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This study was designed to investigate the antigenic cross-reaction which occurs in the macrophage electrophoretic mobility (MEM) assay for lymphocyte sensitiza-tion between encephalitogenic basic protein of myelin (EF), a tumour-specific protein (CaBP), an acid-extractable normal membrane protein (AEMP) and the purified protein derivative of Mycobacterium tuberculosis (PPD). Lymphocytes from multiple sclerosis (MS) and cancer patients have been studied. Cellular affinity chromatography has been used to deplete lymphocyte populations specifically of sensitivity to EF or PPD. Subsequent measurement of the reactivity in the MEM test to the other antigens indicated that: (1) removal of EF sensitized cells was accompanied by a similar reduction in sensitivity to CaBP, AEMP and PPD; (2) removal of PPD sensitized cells gave a similar reduction in sensitivity to EF, but less marked for CaBP and AEMP. This is interpreted as evidence for a sharing of antigenic determinants among these proteins.

Clinical Evaluation of the Macrophage Electrophoretic Mobility Test for Cancer

Lewkonia, R. M.; Kerr, E. J. L.; Irvine, W. J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1974 EN
Relevância na Pesquisa
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Experience with the macrophage electrophoretic mobility (MEM) test of Field and Caspary in subjects with malignant and non-malignant disease is reported. There was some discrimination between groups of patients with benign and malignant lesions but there was no clear separation between the groups. A trial of the Cardiff modification of the test failed to discriminate between groups of patients with benign and malignant chest disease. In the view of the authors the MEM test in its present form is not sufficiently reproducible to warrant more general clinical application as an in vitro test for cancer.

Effect of lymphocyte supernatants on the electrophoretic mobility of erythrocytes: significance in cancer diagnosis.

Dyson, J. E.; Corbett, P. J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1978 EN
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We have determined that when an extract of human brain is preincubated with lymphocytes its subsequent capacity to inhibit the electrophoretic mobility of tanned and stabilized erythrocytes is much reduced. There is a differential effect, however, as the observed reduction is from 73% inhibition to approximately 35% when the pre-incubation is with lymphocytes from patients with malignant disease, but from 73% to approximately 10% when it is with lymphocytes from normal controls. These values were obtained at a brain extract concentration of 333 microgram/ml, with 5 times 10(6) lymphocytes, a pre-incubation time of 18-24 h, and a temperature of 27 degrees C, which are the optimum conditions determined for differentiation between cancer patients and normal subjects. In a series of 73 subjects tested by this method 43/51 cancer patients gave an unequivocal "positive" value, 22/22 normal controls gave a "negative" value, with no false positives.

Lymphocyte supernatants and the electrophoretic mobility of erythrocytes: further experience of cancer diagnosis.

Dyson, J. E.; Watkinson, A. P.; Jones, W. G.; Corbett, P. J.; Joslin, C. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1980 EN
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A double-blind trial of the tanned-erythrocyte electrophoretic mobility test for cancer has been carried out. This included 70 normal subjects as controls, 61 subjects with disease other than cancer, and 229 cancer patients. Slowing values generally increased in the order given, with certain diseases having values within the range positive for cancer. Exposure to viral infection also tended to produce false positives. Slowing values above 50%, however, appear to be definitely associated with cancer. For the middle range of slowing values (25-50%) there is some overlap between the 3 groups, so that a statistical probability of the presence of malignancy is available from the test. With slowing values below 25% there is little likelhood of cancer. Tumour type influences the test result, as does, to a lesser extent, tumour bulk.

Macrophage electrophoretic mobility (MEM) with myelin basic protein.

Rawlins, G. A.; Wood, J. M.; Bagshawe, K. D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1976 EN
Relevância na Pesquisa
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Lymphocytes from a total of 161 subjects, including normal controls and patients with malignant and non-malignant conditions, have been investigated for their response to myelin basic protein, using the macrophage electrophoretic mobility (MEM) test. It has been confirmed that there was a high level of association between clinically evident cancer and a positive response. Lymphocytes from 24/25 patients with non-malignant inflammatory and ischaemic diseases also gave positive responses. In 46 patients with breast lumps studied before mastectomy or biopsy, the test was positive in 15/19 cases which proved to be malignant and in 5/27 which proved benign on histological examination. In its present form the test is not sufficiently reliable for the diagnosis of early cancer. Our results suggest that tissue necrosis in malignant and non-malignant conditions may be one of the factors resulting in sensitization to antigenic determinants present in preparations of myelin basic protein. Despite its technical difficulties, the test may provide a means of examing some aspects of immune recall not readily revealed by other test systems.

The Electrophoretic Mobility of BP8 Ascites Tumour Cells and Allergized Lymph-node Cells After Treatment with Inflammatory Mediators, Ptomaines, Polyamines, Antisera and Neuraminidase or Heparin

Mitchell, D. M.; Cater, D. B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1971 EN
Relevância na Pesquisa
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The electrophoretic mobility (EPM) of BP8 cells was reduced by 5-HT 175 μg./106 cells or 7·5 μg. bradykinin, but 100 μg. histamine was without effect. Cadaverine di HCl or putrescine di HCl 1000 μg./106 cells and agmatine sulphate or tyramine HCl (3000 μg.) reduced the EPM, but spermine tetra HCl was effective at 200 μg. However, 5 μg./106 cells of the polyamines—poly-l-lysine, poly-d-lysine, poly-l-arginine reduced the EPM of BP8 cells to zero, and with larger doses the cells moved towards the cathode. With protamine sulphate the reduction of EPM varied with log dose and 150 μg. did not reduce the EPM to zero. Heparin (3 i.u.) always completely reversed the effects of polyamines or protamine. Lysine HCl reduced the effect of poly-l-lysine. Poly-l-lysine (2 μg.) or poly-l-arginine (4 μg.) divided allergized lymph-node cells (LNC) from C57 B1 mice into 2 populations one still moving towards the anode and the other moving towards the cathode. Treatment of BP8 cells or allergized LNC with neuraminidase reduced their EPM and increased their sensitivity to poly-l-lysine in the presence of Ca++ but reduced it in the absence of Ca++. Heparin reversed the effect of neuraminidase.

A Method of Separating the Lymph-Node Cells, of C57 BL Mice Allergized with BP8 Ascites Tumour, into Two Fractions with a Fast and Slow Electrophoretic Mobility: Demonstration that only the “Fast LNC” will Protect C3H Mice Against Challenge with the Tumour

Thomas, Catherine B.; Cater, D. B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1972 EN
Relevância na Pesquisa
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Lymph-node cells (LNC) from C57 Bl mice, allergized with BP8 ascites tumour, were separated in vitro into 2 populations on the basis of the charge on their plasma membranes. The population with a lower electrophoretic mobility (“slow LNC”) were agglutinated at zero zeta potential by a critical dose of poly-l-lysine leaving the “fast LNC” in suspension. Only the “fast LNC” protected C3H mice against fatal challenge with BP8 tumour in vivo.

Analysis of factor interactions with RNA polymerase II elongation complexes using a new electrophoretic mobility shift assay

Cheng, Bo; Price, David H.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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The elongation phase of transcription by RNA polymerase II (RNAP II) is controlled by a carefully orchestrated series of interactions with both negative and positive factors. However, due to the limitations of current methods and techniques, not much is known about whether and how these proteins physically associate with the engaged polymerases. To gain insight into the detailed mechanisms involved, we established an experimental system for analyzing direct factor interactions to RNAP II elongation complexes on native gels, namely elongation complex electrophoretic mobility shift assay (EC-EMSA). This new assay effectively allowed detection of interactions of TFIIF, TTF2, TFIIS, DSIF and P-TEFb with elongation complexes generated from a natural promoter using an immobilized template. As an application of this assay system, we characterized the association of transcription elongation factor DSIF with RNAP II elongation complexes and discovered that the nascent transcript facilitated recruitment of DSIF. Examples of how the system can be manipulated to address different questions are provided. EC-EMSA should be useful for further investigation of factor interactions with RNAP II elongation complexes.

Polar Residues in Transmembrane Helices can Decrease Electrophoretic Mobility in Polyacrylamide Gels Without Causing Helix Dimerization

Walkenhorst, William F.; Merzlyakov, Mikhail; Hristova, Kalina; Wimley, William C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as membrane mimetic systems. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding...

ELECTROPHORETIC MOBILITY SHIFT ASSAY OF ZINC FINGER PROTEINS: COMPETITION FOR ZN2+ BOUND TO SP1 IN PROTOCOLS INCLUDING EDTA

Kothinti, Rajendra; Tabatabai, Niloofar M.; Petering, David H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA·protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn2+ and readily competes with zinc-finger peptides for Zn2+ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc-finger protein·DNA adducts. This paper examines the chemistry that permits the detection of zinc-finger·DNA complexes in the presence of EDTA, using Zn3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1·GC1 was shown to depend on three properties: the inertness of Zn-Sp1·GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn3-Sp1 under the conditions of the assay that permit some Zn3-Sp1·GC1 to form. Inquiring about the mechanism of stabilization of Zn3-Sp1 by GC1, EDTA readily reacted with Zn3-Sp1 bound to a non-specific DNA, poly(dI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn3-Sp1 failed to compete for zinc bound in the Zn3-Sp1·GC-1 adduct. On the basis of these and other results indicated that the stability of Zn3-Sp1·GC-1 has a thermodynamic not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
EN
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The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

White, Colin P.; Popovici, Jonathan; Lytle, Darren A.; Adcock, Noreen J.; Rice, Eugene W.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2012 EN
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The electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates was measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here provide new insight for Bacillus anthracis surrogate selection and for attachment/detachment and transport studies.