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PubMed1989

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis.

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).

Cell Wall↗
PubMed1975

Analytical characterization of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in renal disease. Clinical and histopathological correlations.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis is a method allowing the separation of proteins according to their molecular sizes. For this reason this method is rather useful for the classification of proteinurias as tubular, glomerular, or mixed. In the present study a good correlation was observed between the results of urine electrophoresis in sodium dodecyl sulphate-polyacrylamide gel and both clinical and histopathological data from 32 patients with different forms of renal disease.

Acute Kidney Injury↗
PubMed2005

Polyacrylamide gel film immobilized molecular beacon array for single nucleotide mismatch detection.

We reported polyacrylamide gel immobilized molecular beacon array for single nucleotide mismatch detection in this paper. Molecular beacons are oligonucleotide probes fluorescing upon hybridization to their complementary DNA/RNA targets with excellent sensitivity and high selectivity. The specially designed molecular beacon for immobilization contains a 15 base loop sequence with a 5 base pair stem, a polyT (20 bases) spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore, and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the polyT is used to minimize disability caused by 5'-end immobilization. The molecular beacon microarray was fabricated by a pin-based spotting robot and the hybridization was investigated by confocal microscope. A real-time hybridization process at room temperature was registered every minute for 20 min after the target solution was pumped into the hybridization cell. The result indicates that a polyacrylamide film coated glass slide provides an ideal solution-like environment for molecular beacon probes. The potential applications of this kind of molecular beacon array are mutation detection, disease mechanisms, disease diagnostics, etc. in a parallel, cost saving, and label-free detection way.

Acrylic Resins↗
PubMed2006

Self-assembly of well-defined polyacrylamide-polystyrene copolymer on fibrillar clays via ultrasonic-assisted surface-initiated atom transfer radical polymerization.

Well-defined polyacrylamide-polystyrene copolymers were grafted from the fibrillar clay, attapulgite, by a four-step self-assembly process: (i) the gamma-aminopropyltriethoxyl silane was self-assembled onto the surfaces of the attapulgite; (ii) the surface amino groups were amidated with bromoacetylbromide; (iii) the bromo-acetamide modified attapulgite was used as macro-initiator for the surface-initiated atom transfer radical polymerization of styrene with the catalyst of the complex of 1,10-phenanthroline and Cu(I)Br; (iv) the polystyrene grafted attapulgite was then used as macroinitiator for the polymerization of acrylamide. The two steps of the surface-initiated atom transfer radical polymerizations were all conducted under ultrasonic irradiation at room temperature. The product, polyacrylamide-polystyrene copolymers grafted attapulgite, had been characterized with elemental analysis, Fourier transform infrared spectroscopy, Thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy, X-ray diffractometry, and transmission electron microscopy.

Acrylic Resins↗
PubMedDate not supplied

Identification and characterization of species of the family Bacteriodaceae by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was used to compare protein profiles of members of the family Bacteriodacceae. Cell-free extracts were prepared and the protein components separated by multistage polyacrylamide gel electrophoresis. The profiles were distinct and reproducible thus allowing identification of species, subspecies, and minor strain differences. Lyophilization of the cell-free extracts did not alter the major protein components. The results indicate that the techniques used may be useful in conjunction with conventional tests in identification at the species level.

Bacteroidaceae↗
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↗
PubMed1990

Comparison of polyacrylamide gel electrophoresis, an enzyme-linked-immunosorbent assay, and an agglutination test for the direct identification of bovine rotavirus from feces and coelectrophoresis of viral RNAs.

The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the "standard," the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGe, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic "super-short" migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.

Animals↗
PubMed1976

Sequential protein analysis from single identified neurons of Aplysia californica. A microelectrophoretic technique involving polyacrylamide gradient gels and isoelectric focusing.

Electrophoresis in polyacrylamide gradient gels and isoelectric focusing in polyacrylamide gels of capillary size are powerful tools for the analysis of molecular weight and charge properties of small protein samples. This is demonstrated using identified neurons from the abdominal ganglion of the sea hare Aplysia californica. Certain cell-specific peptides, which are considered to be neurosecretory, have been shown to be water soluble when ethylene glycol was employed as a mobilizing agent. Although the mode of action of ethylene glycol is not yet understood, this method may be of value for various extraction procedures. The application of a new staining method that is preferential for separations of sodium dodecyl sulfate-proteins yields information about the charge of water-insoluble proteins which has so far been inaccessible. Preliminary results gained by a small, two-dimensional mapping procedure as well as optical density separation patterns of two different nuclear protein fractionation from a single isolated nucleus outline further possibilties of the microgel techniques.

Animals↗
PubMed1983

Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis.

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.

Animals↗
PubMed1975

Amino terminal residue heterogeneity of bovine and ovine growth hormones as revealed by polyacrylamide gel electrophoresis.

Prolonged electrophoresis (4 to 16 h) of bovine and ovine growth hormones on analytical polyacrylamide gels in Tris-EDTA-borate buffers of pH 7.6 to 8.1 resulted in the separation of alanyl, phenylalanyl, and methionyl terminal polypeptide chains of these hormones similar to that obtained by isoelectric focusing in Ampholine pH gradients. Their relative anodic mobilities were methionyl greater than phenylalanyl greater than alanyl. The mobilities of the respective monomers were inversely related to their isoionic points as determined by isoelectric focusing in Ampholine pH gradients. The resolution of the different monomers is attributed to differences in the dissociation constants of the amino groups of the alanyl, phenylalanyl and methionyl terminal residues. Rat growth hormone, which consists of monomers with only a phenylalanyl amino terminus, did not display heterogeneity on gel electrophoresis. The applicability of employing polyacrylamide gel electrophoresis for the assessing of amino terminal residue heterogeneity is noted.

Alanine↗
PubMed1983

Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin.

A total of 53 different cell lines originating from a variety of mammalian species were cultured in vitro and analysed for the presence of vimentin, employing polyacrylamide gradient slab gel electrophoresis in urea/acetic acid as buffer system. Irrespective of the cell culture conditions, and the growth potential and morphology of the cells, vimentin was expressed in all cell lines examined, with two exceptions: MPC-11 mouse myeloma and MOPC-31C mouse plasmacytoma cells. Immunoblotting with the monoclonal antibody alpha-IFA, which is directed against an antigenic determinant shared by all classes of intermediate filaments, did not detect any other of the known intermediate filament proteins in MPC-11 and MOPC-31C cells. Vimentin synthesized by various cell lines was characterized by four different criteria: (1) its extractability with Triton X-100 under various ionic conditions; (2) its behaviour in (NH4)2SO4 fractionation of cellular extracts; (3) its electrophoretic mobility in polyacrylamide gel electrophoresis in urea/acetic acid; and (4) the co-isolation of polypeptides of higher electrophoretic mobility, which, by comparison with degradation products of vimentin obtained with the Ca2+-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca2+-dependent proteolysis of vimentin. Although the degradation products occurred in different ratios in extracts of different cell lines, they constituted the same characteristic set of proteins whenever degradation of vimentin was observed. The formation of proteolytic breakdown products could be partially to totally suppressed when the cells were harvested, washed and processed in the presence of EGTA and proteinase inhibitors. The experimental data show that: (1) vimentin, as well as the Ca2+-activated proteinase specific for intermediate filament proteins, is highly conserved during the evolution of mammalian species; (2) the proteolytic breakdown products of vimentin, which give rise to a characteristic 'staircase' in two-dimensional gel electrophoresis, are probably artefacts of isolation; (3) the expression of vimentin is neither a prerequisite for nor necessarily indicative of rapid cell proliferation in vitro; and (4) the techniques described can be used for the routine identification of vimentin in cells and tissues in case vimentin-specific antibodies are not available.

Animals↗
PubMed1992

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analyses of ureaplasmas isolated from dogs.

To define the antigenic relatedness among unspeciated ureaplasma strains isolated from dogs, four canine ureaplasma isolates representing four serotypes were compared with the five type strains of the established species in Genus Ureaplasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Although all the strains showed distinct electrophoretic patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the Western blotting patterns were much more distinct. By Western blotting, the five type strains of established species reacted strongly with homologous antisera and showed slight cross reactions with heterologous antisera. However canine strains which showed little cross reactions with the established Ureaplasma species showed a variety of cross reactions among the four canine serotypes used.

Animals↗
PubMed1989

Detection of IgG oligoclonal bands in unconcentrated CSF by isoelectric focusing in ultrathin polyacrylamide gel, direct antiserum immunofixation and silver nitrate staining.

Isoelectric focusing of proteins in ultrathin polyacrylamide gel (0.4 mm), followed by direct immunofixation with monospecific antisera and silver nitrate staining, is a highly specific, sensitive and simple method for the detection of oligoclonal IgG in unconcentrated CSF samples. The ultrathin polyacrylamide gels have several advantages, i.e. significantly smaller amounts of reagents are required, and thinner gel can be more efficiently cooled, resulting in higher resolution and shorter running, washing, staining and destaining times. Direct immunofixation in the gel, a time-saving and simple step, increases the sensitivity and specificity of the method. We reduced the samples to 5-10 microliters. For the present method, the optimal concentration of IgG was 0.025-0.030 g/l. It is possible to detect oligoclonal IgG bands at an IgG concentration corresponding to the applied amount of 80-100 ng. In our testing of this method, oligoclonal bands in CSF specimens were clearly demonstrated in 33 (97%) out of 34 patients with definite multiple sclerosis, in 16 (42%) out of 38 patients with infectious diseases of the central nervous system and in 11 (18%) out of 58 patients with other neurological disorders. The method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in unconcentrated CSF samples, and can be recommended for use in the CSF laboratory routine.

Central Nervous System Diseases↗
PubMed1997

Simplified sizing of low-density lipoprotein using polyacrylamide gradient gel electrophoresis of plasma.

Low-density lipoprotein (LDL) particles can be separated into subfractions according to size by non-denaturing polyacrylamide gradient gel electrophoresis. Established research methods require specialised equipment and are frequently unsuited to the clinical laboratory. In this study, we utilised a colour flat bed scanner in conjunction with shareware image analysis software to compare LDL particle diameters of isolated LDL with LDL in whole plasma. LDL was isolated by ultracentrifugation and electrophoresed on 3-13% gels (Gradipore; Sydney, Australia) for 2400 Volt-hours in parallel with plasma and molecular size standards. Coomassie Blue-stained gels were scanned in reflexive mode using a colour flat-bed scanner and Adobe Photoshop 3.0 software. Density traces of each lane were obtained using NIH Image software (public domain, USA). LDL particle diameters were determined from calibration curves of the log of molecular diameter of standards against migration distance. There was a good correlation between LDL particle diameters obtained using isolated LDL and whole plasma (r = 0.87, P < 0.001; n = 22). However, the group means (+/- S.D.) (24.7 +/- 0.6 and 24.8 +/- 0.5 nm respectively) were statistically different on the paired t-test (P < 0.05). It is unclear whether this numerically small difference is due to alterations in LDL during the longer preparative procedures for LDL, or to matrix effects during electrophoresis of plasma samples. In conclusion, plasma samples stained with Coomassie Blue and scanned with a colour flat bed scanner can conveniently be used for LDL particle sizing by non-denaturing polyacrylamide gradient gel electrophoresis.

Adult↗
PubMedDate not supplied

Acrylamide and polyacrylamide: a review of production, use, environmental fate and neurotoxicity.

Acrylamide is a highly water soluble vinyl monomer formed from the hydration of acrylonitrile. The major commercial use of acrylamide is the formation of polymers. In the environment acrylamide has a high mobility in soil, may travel great distances in ground-water, is biodegradable, and is not absorbed by sediments or affected by water treatment. It is absorbed by all routes of animal exposure. The main metabolite is N-acetyl-S-(3-amino-3-oxypropyl)-cysteine and is excreted predominantly in the urine. Acrylamide produces an ascending central/peripheral axonopathy in man and animals. The major histological findings are swelling of axons and/or decrease in number of large diameter axons. Acrylamide axonopathy is reversible with time, but full recovery depends upon the severity of the intoxication. All reported cases of acrylamide toxicity have been attributed to handling the monomer. Polyacrylamide is non-toxic. Specific clinical features of acrylamide intoxication are more conclusive than electrophysiological, histological or biochemical laboratory tests for diagnosis. Acrylamide can be detected by titration, colorimetry, high performance chromatography, gas chromatography and polarography in air, water, biological fluids, tissues and polyacrylamides. Present research on the effects of acrylamide focuses on developmental and reproductive effects, genotoxicity and carcinogenicity.

Acrylamides↗
PubMedDate not supplied

The electrophoretic separation of curved cisplatin-modified DNA fragments on polyacrylamide gels is dependent on the voltage gradient.

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The parameters of the electric field do not affect separation of 'normal' DNA fragments of comparable length.

Base Sequence↗
PubMed1976

A comparative study of preparations of ovine lutenizing hormone by bioassay, immunodoublediffusion, radioimmunoassay, radioreceptor assay and polyacrylamide gel electrophoresis.

Three preparations of ovine LH were compared for biological potency and by several in vitro parameters. All were found to be heterogenous by immunodoublediffusion and by electrophoresis in polyacrylamide gels. They all also showed similarities and/or differences with respect to their characteristics in immunodoublediffusion, radioimmunoassay, radioreceptor assay, gel electrophoresis and in dye-binding capacity, but in ways that preclude establishing a meaningful correlation between biopotency and the in vitro parameters or even among the in vitro parameters themselves. The implications of these findings for the use of these in vitro parameters for screening and assessing biological potencies of LH preparations and for inferring chemical and/or structural similarities between LH preparations are discussed. Aspects of polymorphism of LH, observed by electrophoresis on polyacrylamide gels, are also discussed.

Animals↗
PubMed1978

Separation of relaxin activities in extracts of ovaries of pregnant sows by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis has been employed to separate three fractions that have relaxin activity from crude extracts of ovaries of pregnant sows. Bioassays indicated that all fractions had the ability to inhibit spontaneous uterine contractions in vitro and to induce formation of interpubic ligaments in oestrogen-primed mice. The presence of the various fractions in crude extracts of sow ovaries was monitored with the polyacrylamide gel system on days 40, 70 and 100 of pregnancy. Both the total amount and the relative proportions of the fractions were found to change as pregnancy progressed.

Animals↗