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At least 19 recordsLinked to original sources

Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗

Solid-phase vitamin B12 assays using polyacrylamide-bound intrinsic factor and polyacrylamide-bound R-binder.

A new solid phase vitamin B12 assay is described using intrinsic factor to measure microbiologically-available B12 and R-binder to measure total B12. The solid phase reagent consists of intrinsic factor coupled to polyacrylamide beads and salivary R-binder coupled to polyacrylamide beads. The assay is simple to perform and separates completely sera from controls and patients with megaloblastic anaemia due to B12 deficiency.

Acrylic Resins↗

Sodium dodecyl sulfate-polyacrylamide solution-filled capillary electrophoresis of proteins using stable linear polyacrylamide-coated capillary.

Sodium dodecyl sulfate (SDS)-linear polyacrylamide solution-filled capillary electrophoresis using a stable coated fused silica capillary has been developed for the separation and molecular weight determination of proteins. A fused silica capillary was coated with linear polyacrylamide through Si-C bond which suppressed the electro-osmotic flow and reduced the adsorption of proteins. Acrylamide solution containing SDS was filled into the coated capillary and polymerized. Compared with a conventional fused silica capillary coated with 3-methacryloxypropyltrimethoxysilane through siloxane linkage (Si-O-Si-C), the developed capillary was much more stable, even in the alkaline condition. Consequently, the durability of the polymer matrices and the reproducibility of separation were markedly improved. An excellent linear relationship was obtained between the mobility and the logarithm of the protein molecular weight. The relative standard deviation of migration times was below 1%.

Electrophoresis, Polyacrylamide Gel↗

Analytical gradient polyacrylamide gel-crossed line immunoelectrophores. A technique for locating species specific Mycobacterium tuberculosis H37RV antigens in polyacrylamide gel columns.

Analytical gradient polyacrylamide gel electrophoresis of antigens from Mycobacterium tuberculosis H37RV was combined with crossed line immunoelectrophoresis to locate the position of antigens in polyacrylamide gel columns that appeared to be specific for M. tuberculosis H37RV. Specificity was established by comparing H37RV with M. kansaii CE in the electrophoretic procedure and the relative mobility (Rm) values are being used in an effort to recover and enrich specific antigens from preparative gel columns. Such fractions should be more suitable in efforts to recover purified monospecific components from M. tuberculosis H37RV.

Antigens, Bacterial↗

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

Chrysosporium↗

Fabric-reinforced ultrathin polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing.

The suitability of four different fabric materials for the preparation of ultrathin rein-forced polyacrylamide gels was investigated. With all fabric-reinforced gels, a good separation of proteins by isoelectric focusing and sodium dodecyl sulfate-electrophoresis could be achieved. Semi-dry electrophoretic blotting of proteins was possible with all types of fabric-reinforced gels. Two polyester fabrics (a net and a fleece) were decidedly superior in handling and dimensional stability on drying to a nylon fabric and another polyester fleece material. Only gels prepared with the former materials withstood further treatment, such as fixation, staining, destaining, and drying. One of the polyester fleece fabrics had poor handling properties and the nylon fabric was unsuitable for direct staining procedures employing concentrated (20% w/v) trichloroacetic acid as fixative.

Electrophoresis, Polyacrylamide Gel↗

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamides↗

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin.

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.

Amyloid↗

Chromogenic substrate autography: a method for detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels.

A chromogenic substrate autography is described which allows characterization and quantification of serine proteases in crude systems after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. Separation of samples containing proteases by either method is followed by an overlay of the gels on an indicator film prepared by incorporation of a suitable paranitroanilide substrate into agarose. Positions of the proteases are revealed by the formation of yellow-colored zones which can be quantified by densitometry at 405 nm. The technique proved suitable for determination of molecular weights, isoelectric points, and quantitative measurements of amidolytic activities of urokinase, tissue plasminogen activator, tissue and plasma kallikrein, and thrombin in biological fluids and purified preparations.

Animals↗

Polyacrylamide + Al2(SO4)3 and polyacrylamide + CaO remove coliform bacteria and nutrients from swine wastewater.

Animal wastes are a major contributor of nutrients and enteric microorganisms to surface water and ground water. Polyacrylamide (PAM) mixtures are an effective flocculent, and we hypothesized that they would reduce transport of microorganisms in flowing water. After waste water running at 60.0 1 min(-1) flowed over PAM + Al2(SO4)3, or PAM + CaO in furrows, total coliform bacteria (TC) and fecal coliform bacteria (FC) were reduced by 30-50% at 1 and 50 m downstream of the treatments compared to the control. In a column study, PAM + Al2(SO4)3, and PAM + CaO applied to sandy, sandy loam, loam, and clay soils reduced NH4+ and ortho-P concentrations in leachate compared to the source waste water and the control. PAM + Al2(SO4)3 and PAM + CaO applied to sandy, sandy loam and loam soils reduced both total and ortho-P, concentrations in leachate compared to he source wastewater and control treatment. In a field study, PAM + Al2(SO4)3, or PAM + CaO treatments did not consistently reduce NH4+, NO3-, ortho-P, and total P concentrations in wastewater flowing over any soil compared to inflow wastewater or the control treatment. With proper application PAM + Al2(SO4)3 and PAM + CaO may be able to reduce the numbers of enteric bacteria in slowly flowing wastewater running off animal confinement areas, reducing the amount of pollutants entering surface water and groundwater.

Acrylic Resins↗

The immunogenicity of dinitrophenylated polyacrylamide and the adjuvant effect of polyacrylamide.

Dinitrophenyl-substituted polyacrylamide (PAAm-DNP) is weakly immunogenic in guinea pigs and rats. Only 2-mercaptoethanol-sensitive IgM antibodies are elicited, and no immunological memory in guinea pigs is appreciated. A 15000-fold carrier excess, given 6 days before an immunogenic dose of PAAm-DNP, does not impair the anti-DNP response. Different doses of PAAm failed to increase the level of natural anti-DNP or anti-sulfanilic acid antibodies, either in guinea pigs or rats. Nevertheless, when given together with sheep red blood cells. PAAm increases the anti-sheep red blood cell humoral immune response in rats. It is suggested from the above data that the PAAm-DNP immunogenicity is not accounted for by the 'two-signal' model of B-cell activation (via 'associative antibodies'), nor by a model that ascribes B-cell activation to the polyclonal B-cell activating properties of the carrier. However, the possibility that the adjuvant effect showed by PAAm plays a role in allowing the conjugate PAAm-DNP to be immunogenic, remains an open question, and must be further investigated.

Acrylamides↗