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PubMed1986

The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis.

A simple physical-chemical method for estimating the lipopolysaccharide (LPS, i.e. endotoxin) content of meningococcal polysaccharide vaccines is described. The low molecular weight LPS was first separated from the high molecular weight polysaccharide by SDS-polyacrylamide gel electrophoresis. The LPS was then selectively visualized by a highly sensitive silver stain which can detect 5 ng of meningococcal LPS. The method can detect concentrations of LPS in meningococcal polysaccharides as low as 0.01%, and was used to measure semiquantitatively the LPS in bulk powders of meningococcal polysaccharides and in vaccines in final containers. The LPS in the bulk powders and in the vaccines was also assayed by the gelation of Limulus polyphemus ameobocyte lysate and the results of the two methods were in agreement.

Animals↗
PubMed1999

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility.

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.

Analysis of Variance↗
PubMed1999

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polymerase chain reaction for differentiation of Prevotella intermedia and Prevotella nigrescens.

Isolates previously thought to be Prevotella intermedia have been shown to be a closely related species now known as Prevotella nigrescens. The purpose of this study was to determine the efficacy of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) to differentiate endodontic isolates of P. nigrescens from P. intermedia. Fifty-six strains of black-pigmented bacteria isolated from endodontic infections and conventionally identified as P. intermedia were used in this study. Using SDS-PAGE, novel polypeptide bands were used to differentiate P. nigrescens from P. intermedia. PCR was accomplished with specific primers for the 16S ribosomal RNA gene of both strains. Of 56 endodontic isolates, 41 (73%) strains were identified by SDS-PAGE as P. nigrescens and 15 (27%) strains as P. intermedia. Of the 41 strains of P. nigrescens identified by SDS-PAGE, PCR identified 37 strains as P. nigrescens. Restriction endonuclease digestion of amplified 16S ribosomal RNA genes indicated that the remaining four strains originally identified by SDS-PAGE as P. nigrescens were actually strains of Prevotella distinct from P. nigrescens and P. intermedia. Of 15 strains of P. intermedia identified by SDS-PAGE, PCR identified 14 strains as P. intermedia; but, one strain was identified as P. nigrescens. The results indicated that PCR was a more precise method than SDS-PAGE to differentiate P. intermedia from P. nigrescens. This study confirms that P. nigrescens is more commonly isolated in pure culture from endodontic infections than P. intermedia.

Bacterial Typing Techniques↗
PubMed2003

Microstructural modifications induced by the entrapped glucose oxidase in cross-linked polyacrylamide microgels used as glucose sensors.

Glucose oxidase (GO(x)) has been immobilized in cross-linked polyacrylamide microgels using the concentrated emulsion polymerization method. The immobilization of the enzyme is effective when the number of cross-links is equal or greater than 0.021mol/g. The swelling of the microgels is not affected by the entrapped enzyme but decreases with increasing the number of cross-links. The glass transition temperature of microgels with GO(x) presents considerable differences with that of the empty microgels, which are explained on base of the competition between the plasticizer effect of the encapsulated enzyme and the chain mobility restrictions imposed by the microgel cross-linking content. Immobilization of GO(x) slightly modifies the X-ray diffraction patterns of the microgels; however, the diffraction profiles of cross-linked microgels suggest the formation of bis(acrylamide) aggregates in the outer layers of the microgel particles.

Acrylic Resins↗
PubMed2001

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.

Cerebrospinal Fluid Proteins↗
PubMed2002

2-D gel densitometer for high-contrast and selective imaging of chlorophyll-containing protein complexes separated by non-denaturing polyacrylamide gel electrophoresis.

In this work, we present a home-made two-dimensional (2-D) CCD imaging system for the monochromatic densitometry of plane gels and its application to the imaging and densitometry of chlorophyll (Chl)-containing proteins separated by non-denaturing polyacrylamide gel electrophoresis. The monochromatic imaging of separated green bands at the wavelengths corresponding to their absorption band increases their contrast. This allows a better visualization of the faint-green bands in the gel and using of samples with lower Chl content for the electrophoresis. By the comparison of 2-D densitograms of the same gel illuminated with 670 and 650 nm lights, that is, at the red absorption maximum of Chl a and b, respectively, we achieved a selective imaging of the complexes with different Chl a/b ratio. This approach was used to specify an unknown band that appeared in the gel of the sample prepared from the thylakoid membranes of preheated barley leaves.

Chlorophyll↗
PubMed1996

A fluorogenic substrate for beta-glucuronidase: applications in fluorometric, polyacrylamide gel and histochemical assays.

We have developed a fluorogenic substrate, ELF-97 beta-D-glucuronide, that provides significant advantages over existing substrates in detecting beta-glucuronidase activity. ELF-97 beta-D-glucuronide allows the detection of enzymatic activity in situ, yielding a hydrolytic product that exhibits maximal fluorescence within the physiological pH range. This substrate yields a hydrolytic product that demonstrates a more than 100 nm Stokes shift, which minimizes interference from autofluorescence in plant tissue. With the commercial enzyme, ELF-97 beta-D-glucuronide can detect less than 2 x 10(-4) U/ml of beta-glucuronidase activity in solution, and 5 x 10(-4) U per lane in polyacrylamide gels. Assays using transgenic Arabidopsis, whole leaf extracts of GUS-positive, but not GUS-negative plans, show significant GUS activity upon incubation with ELF-97 beta-D-glucuronide. Furthermore, the ability of this substrate to form insoluble precipitates at the sites of enzymatic activity makes it suitable for in situ localization of GUS activity in tissue samples of higher plants.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Polyphenol oxidase activity staining in polyacrylamide electrophoresis gels.

An analytical method allowing the detection of polyphenol oxidase activity on polyacrylamide gel electrophoresis (PAGE) is described. The method is rapid, sensitive and specific and is based on a coupling reaction between 4-tert-butyl-o-benzoquinone and the aromatic amine, 4-amino-N,N-diethylaniline sulphate. Catecholase activity of polyphenol oxidase appears as blue stained bands on a colourless background.

Animals↗
PubMed1999

Analysis of viral glycoproteins by glycosidic digestion inside a polyacrylamide gel.

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.

Animals↗
PubMed2000

Silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus Trametes.

Silver stained denaturing polyacrylamide gels (PAGEs) and fluorescent denaturing automated capillary electrophoresis (CE) were used to detect amplified fragment length polymorphism (AFLP) patterns obtained from white-rot fungi belonging to the genus Trametes. AFLP fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of Trametes ochracea, nine strains of Trametes hirsuta and ten isolates of Trametes versicolor. UPGMA dendrograms derived from fluorescently labelled and silver stained AFLP patterns were similar, but a few differences were detected especially in the clustering of T. ochracea and T. hirsuta strains. Compared to silver-stained AFLP, detection of fluorescent AFLP was fast, reliable and easy to perform and it facilitated surveying with a computerized analysis system. Fluorescent CE seems to be well suited for studying similarity between Trametes species.

Basidiomycota↗
PubMed1999

Detection of beta-1,6-glucanase isozymes from Trichoderma strains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing gels.

The filamentous fungus Trichoderma produces, under specific growth conditions, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the antagonistic action of Trichoderma against a wide range of fungal plant pathogens. In this report we describe two different methods for the specific detection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase activity can be assayed in the gel by renaturation of the enzyme, incubation with an overlay agarose gel containing solubilized pustulan (a commercially available beta-1,6-glucan), followed by the staining of the agarose gel with Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be detected after incubation with solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium chloride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under different growth conditions.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Resolution of high-molecular-weight components in lipopolysaccharides of Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus strains with sodium dodecyl sulfate polyacrylamide gels.

The use of 0.5% sodium dodecyl sulfate in polyacrylamide separation gels allowed the resolution in several bands of high-molecular-mass components in smooth lipopolysaccharide of bacterial outer membrane from Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus. With or without 0.1% SDS, however, such a result was not possible.

Citrobacter↗
PubMed2001

Chemically modified polyacrylamide-g-guar gum-based crosslinked anionic microgels as pH-sensitive drug delivery systems: preparation and characterization.

New spherically shaped cross-linked hydrogels of polyacrylamide-grafted guar gum were prepared by the emulsification method. These were selectively derivatized by saponification of the -CONH2 group to the -COOH group. The derived microgels were characterized by FTIR and elemental analyses. The derivatized microgels were responsive to pH and ionic strength of the external medium. The swelling of microgels increased when the pH of the medium changed from acidic to alkaline. Transport parameters, viz., solvent front velocity and diffusion coefficients were calculated from a measurement of the dimensional response of the microgels under variable pH conditions. The variation in pH changed the transport mechanism from Case II (in 0.1 N HCl) to non-Fickian (in pH 7.4 buffer), and these processes are relaxation-controlled. Ionic strength exerted a profound influence on the swelling of the microgels. Swelling was reversible and pulsatile with the changing environmental conditions. The pH-sensitive microgels were loaded with diltiazem hydrochloride and nifedipine (both antihypertensive drugs) and their release studies were performed in both the simulated gastric and intestinal pH conditions. The release was relatively quicker in pH 7.4 buffer than observed in 0.1 N HCl; the release followed non-Fickian transport in almost all the cases.

Acrylic Resins↗
PubMed1982

The use of TNP-conjugated polyacrylamide beads in a C1q binding inhibition test for circulating immune complexes.

A new assay for the detection of circulating immune complexes is described. It is based on the same principle as the C1q deviation test: the binding of radiolabelled C1q to a solid phase is inhibited by immune complexes. Trinitrophenylated polyacrylamide beads are used as a stable C1q-reactive solid phase in our test. Aggregated IgG in normal serum could be detected by this method to a minimum concentration of about 10 microgram/ml. The test was used to quantitate circulating immune complexes in sera of patients with glomerulonephritis, liver diseases, lymphoma, and myeloid leukemia. The results are compared and correlated with those obtained by the C1q binding assay for the same sera.

Acrylic Resins↗
PubMed1981

Peptide mapping of collagen chains using CNBr cleavage of proteins within polyacrylamide gels.

A peptide mapping technique is described which uses a two dimensional format to display CNBr peptides of collagen chains. Biosynthetically-labeled products from 250,000 cells are analyzed in each map by a rapid procedure which does not require preliminary purification steps. Proteins trapped within polyacrylamide gels are digested with CNBr under conditions where diffusion of radiolabeled peptides from gels is negligible, and the reaction products are recovered quantitatively by electroelution. Peptide maps of pro alpha chains, alpha chains, and TCA chains cleaved with mammalian collagenase are presented with the identification of specific fragments. This method is useful for the analysis of structural abnormalities in collagen proteins from patients with certain genetic disorders, examination of collagenous proteins produced by primary cultures which exhibit phenotypic switching, and identification of new collagen types.

Cells, Cultured↗
PubMed1984

The alpha 1(I) and alpha 2(I) chains of collagen separate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to differences in sodium dodecyl sulfate binding capacities.

The alpha 1(I) and alpha 2(I) chains of rat tail collagen, indistinguishable with respect to their chain lengths, are well separated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). In the present study, the results demonstrated: 1) the alpha 2 chain maximally binds SDS in an amount of 1.6 g/g which is significantly larger than 1.4 g/g found for the alpha 1 chain; 2) the alpha 2 chain acquires electrophoretic mobility in a free solution 14% higher than that of the alpha 1 chain as the result of the larger amount of SDS bound; 3) although both of the chains have virtually the same hydrodynamically effective size, the above difference in mobility persists in a gel, and brings about the separation in a high resolution. It was also found that the alpha 2 chain binds appreciable amounts of SDS in the initial phase of the binding where the alpha 1 chain hardly binds SDS.

Animals↗
PubMed1987

Ascorbate effects on type I procollagen synthesis by human adult skin fibroblasts: different migration positions of type I procollagen chains on SDS polyacrylamide gel after incubation with ascorbate.

The effects of ascorbate and steroids on type I procollagen synthesis by human skin fibroblasts were studied. Ascorbate treatment (50 micrograms/ml) for 24 hours stimulated a 2-3 fold increase in type I procollagen synthesis and an unexpected shift in the mobility of type I procollagen on SDS polyacrylamide gels. The kinetics of the increase in procollagen synthesis (4 hours) and the shift in electrophoretic mobility (1 hour) were dissimilar, suggesting different controlling mechanisms. This was confirmed by the addition of alpha-alpha'-dipyridyl to ascorbate-treated cultures which eliminated the ascorbate-induced shift in electrophoretic mobility without altering the amount of procollagen synthesis. In contrast, hydrocortisone (1.5 microM) reduced the ascorbate-induced stimulation of type I procollagen synthesis by 80% but did not affect the ascorbate-induced shift in electrophoretic mobility. These studies indicate that the ascorbate-induced increase in type I procollagen synthesis is due to increased levels of type I procollagen mRNA and is independent of the level of hydroxylation of the procollagen.

2,2'-Dipyridyl↗
PubMed2002

Polyacrylamide preparations for protection of water quality threatened by agricultural runoff contaminants.

Waste streams associated with a variety of agricultural runoff sources are major contributors of nutrients, pesticides and enteric microorganisms to surface and ground waters. Water soluble anionic polyacrylamide (PAM) was found to be a highly effective erosion-preventing and infiltration-enhancing polymer, when applied at rates of 1-10 g m(-3) in furrow irrigation water. Water flowing from PAM treated irrigation furrows show large reductions in sediment, nutrients and pesticides. Recently PAM and PAM + CaO and PAM + Al(SO4)3 mixtures have been shown to filter bacteria, fungi and nutrients from animal wastewater. Low concentrations of PAM [175-350 g PAM ha(-1) as PAM or as PAM + CaO and PAM + Al(SO4) mixture] applied to the soil surface, resulted in dramatic decreases (10 fold) of total, coliform and fecal streptococci bacteria in cattle, fish and swine wastewater leachate and surface runoff. PAM treatment also filtered significant amounts of NH4, PO4 and total P in cattle and swine wastewater. This points to the potential of developing PAM as a water quality protection measure in combination with large-scale animal feeding operations. Potential benefits of PAM treatment of animal facility waste streams include: (1) low cost, (2) easy and quick application. (3) suitability for use with other pollution reduction techniques. Research on the efficacy of PAM for removal of protozoan parasites and viruses and more thorough assessment of PAM degradation in different soils is still needed to completely evaluate PAM treatment as an effective waste water treatment. We will present analysis and feasibility of using PAM, PAM + Al(SO4)3, and PAM + CaO application for specific applications. Our results demonstrate their potential efficacy in reducing sediment, nutrients and microorganisms from animal production facility effluents.

Acrylic Resins↗