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PubMed1989

Study of grasshopper meiosis-associated proteins using two-dimensional polyacrylamide gel electrophoresis.

Premeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One-dimensional sodium dodecyl sulphate-polyacrylamide gels detected differences between premeiotic and meiotic samples but two-dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis-associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.

Animals↗

PubMed1992

Why can we not sequence thousands of DNA bases on a polyacrylamide gel?

Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field-induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high-field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.

Acrylic Resins↗

PubMed1996

Eliciting macroporosity in polyacrylamide and agarose gels with polyethylene glycol.

Preparation of highly porous polyacrylamide has recently been described (Righetti et al., Electrophoresis, 1992, 13, 587-595). In this report we add new observations on the conditions of promoting macroporosity in polyacrylamide gels and extend the possibility of eliciting this phenomenon to agarose matrices by the combined use of polyethylene glycol and glycerol. The process of cluster formation in hot agarose solutions was studied and gel structures were examined by scanning electron microscopy. A definition of macroporosity in gel, related to controlled microsyneresis during gelation, is tentatively proposed. The unexpected influence of acrylamide and agarose concentrations upon the size of macroporous structures in the corresponding gels is revealed.

Acrylic Resins↗

PubMed1996

Identification of different galectins by immunoblotting after two-dimensional polyacrylamide get electrophoresis with immobilized pH gradients.

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

Brain Chemistry↗

PubMed1997

Oriented macroporous polyacrylamide gels.

Macroporous gels with huge cavities and partition walls result from controlled microsyneresis during gelation. In this report we show that the microsyneresis process can be further controlled: it is possible to orient the partition walls of macropores by the use of an electric field throughout polymerization. Our oriented macroporous polyacrylamide gels offer a specific structure: a set of parallel channels of about 2 microm inner diameter, with polyacrylamide walls including acidic groups linked to the aggregated matter.

Acrylic Resins↗

PubMed1998

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis.

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.

Blood Proteins↗

PubMed2005

Efficient and sensitive method of DNA silver staining in polyacrylamide gels.

DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively.

Acrylic Resins↗

PubMed1977

Reactions of human platelets with microspheres of poly(hydroxymethyl methacrylate) and polyacrylamide.

We have studied the contact interaction of platelets with hydrogels. In the form of microspheres, 0.6-1.0 mu, poly(glycol methacrylate) (polyHEMA) and poly(methyl methacrylate) beads cause platelets to aggregate at concentrations of about 10(8) beads/ml. Polyacrylamide and (20/80) poly(acrylamide-HEMA) copolymer were ineffective in aggregating platelets. The admixture of 20% methacrylate to polyHEMA rendered the beads inactive. Blood plasma components other than fibrinogen were found essential to the interaction of the beads with platelets. Near-infrared spectra of the hydrogels polyacrylamide and polyHEMA showed the water hydrogen bonds to be the same for both and different from those in pure water. The monomer HEMA is an inhibitor of platelet aggregation and the release reaction at levels of 0.1%. It is concluded that the two hydrogels have different blood compatibilities, which depend more on the network structures than the water structures in the respective gels.

Acrylamides↗

PubMedDate not supplied

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding.

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.

Acrylic Resins↗

PubMed2004

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping.

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.

Alkanesulfonates↗

PubMed1991

Two-dimensional polyacrylamide gel electrophoresis characterization of APz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins.

A boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two-dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm-egg binding and bound heat-solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm-egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm-egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat-solubilized ZP was added to sperm at doses that block sperm-egg binding and the excess ZP was removed, the sperm-egg blocking activity of the antibody was not absorbed by these sperm, and antibody-containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm-egg blocking activity again was not absorbed by antibody-treated cells. Beta ZP3, however, failed to block sperm-egg binding and failed to absorb the sperm-egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.

Animals↗

PubMed1993

High resolution polyacrylamide gel electrophoresis of Physarum polycephalum histones: improved resolution of variants and modified forms.

An electrophoresis system providing a good resolution of all basic proteins and their variants or modified forms extracted from Physarum polycephalum nuclei was developed. This two-dimensional polyacrylamide gel electrophoresis system combines the use of an AUT gel containing a preformed gradient of urea in the first dimension and a SDS polyacrylamide gel in the second one.

Animals↗

PubMed1998

Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips.

Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino-DNA and amino-oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

Acrylic Resins↗

PubMed1999

The Effect of Polymer Substrate Surface Basicity on the Adsorption of a Cationic Polyacrylamide.

The adsorption of a cationic polyacrylamide onto polytetrafluoroethylene, bisphenol-A based epoxy resins, and acrylate substrates was characterized with X-ray photoelectron spectroscopy (XPS) and dynamic contact angle measurement in water. Surface acidity-basicity was quantified via dynamic contact angle measurement in water, ethylene glycol, and diiodomethane. The epoxy resins exhibited intermediate surface basicity and adsorbed the most polyelectrolyte, suggesting that specific substrate-adsorbate acid-base interactions govern adsorption of the cationic polyacrylamide onto surfaces of low to intermediate basicity. In general polar interactions appear to dominate the adsorption process. Advancing contact angle measurements have yielded quantitative fractional surface coverage information for polymer-on-polymer adsorption comparable to that obtained via XPS. Copyright 1999 Academic Press.

Journal Article↗

PubMed2001

Spontaneous Vesicle Formation in Aqueous Mixtures of Cationic Surfactants and Partially Hydrolyzed Polyacrylamide.

Spontaneous vesicle formation was observed in aqueous mixtures of partially hydrolyzed polyacrylamide and single-tailed cationic surfactants. The influences of pH, addition of long-chain alcohols, variation of hydrocarbon chain length, and head group size of the cationic surfactants were investigated systematically. It was found that 30-50% hydrolyzed degree of polyacrylamide and cationic surfactants with 10 or 12 methylene groups are suitable for vesicle formation in the mixed aqueous systems at pH 9.2. Addition of long-chain alcohols was found to be helpful for vesicle formation in some cases. The vesicle stability was also investigated at various temperatures and amounts of ethanol addition. Copyright 2001 Academic Press.

Journal Article↗

PubMed2002

Kinetics of adsorption of high molecular weight anionic polyacrylamide onto kaolinite: the flocculation process.

The adsorption kinetics of anionic polyacrylamide flocculant onto kaolinite clay are examined as a function of flocculant dosage and pH. Special attention has been given to the flocculation effect during the adsorption process and the resulting inhibition of further adsorption. At pH 8.5 the adsorption capacity of anionic polyacrylamide on kaolinite is low while at pH 4.5, the adsorption capacity increases. Flocculant adsorption has been shown to be related to the amount of available surface area, pH, flocculant dosage, and the resulting floc strength, which controls the rate of new surface area exposure and hence the continuation of further adsorption. At both pH 4.5 and pH 8.5, complete adsorption is achieved at low flocculant dosages and adsorption equilibrium is achieved at high flocculant dosages after 1 day. In contrast, at intermediate flocculant dosages adsorption equilibrium is not reached over a 7-day period, due to a continuously increasing surface area.

Journal Article↗

PubMed1998

Proton Diffusion and T1 Relaxation in Polyacrylamide Gels: A Unified Approach Using Volume Averaging.

The structure of polyacrylamide gels was studied using proton spin-lattice relaxation and PFG diffusion methods. Polyacrylamide gels, with total polymer concentrations ranging from 0.25 to 0.35 g/ml and crosslinker concentrations from 0 to 10% by weight, were studied. The data showed no effect of the crosslinker concentration on the diffusion of water molecules. The Ogston-Morris and Mackie-Meares models fit the general trends observed for water diffusion in gels. The diffusion coefficients from the volume averaging method also fit the data, and this theory was able to account for the effects of water-gel interactions that are not accounted for in the other two theories. The averaging theory also did not require the physically unrealistic assumption, required in the other two theories, that the acrylamide fibers are of similar size to water molecules. Contrary to the diffusion data, T1 relaxation measurements showed a significant effect of crosslinker concentration on the relaxation of water in gels. The model developed using the Bloch equations and the volume averaging method described the effects of water adsorption on the gel medium on both the diffusion coefficients and the relaxation measurements. In the proposed model the gel medium was assumed to consist of three phases (i.e., bulk water, uncrosslinked acrylamide fibers, and a bisacrylamide crosslinker phase). The effects of the crosslinker concentration were accounted for by introducing the proton partition coefficient, Keq, between the bulk water and crosslinker phase. The derived relaxation equations were successful in fitting the experimental data. The partition coefficient, Keq, decreased significantly as the crosslinker concentration increased from 5 to 10% by weight. This trend is consistent with the idea that bisacrylamide tends to form hydrophobic regions with increasing crosslinker concentration. Copyright 1998 Academic Press.

Journal Article↗

PubMed1979

Characterization of alpha-1-antitrypsin by isoelectric focusing on an ultrathin polyacrylamide gel layer. An economic high-resolution system for determining PiM subtypes.

Through the use of ultrathin layer polyacrylamide gel isoelectric focusing it is possible to obtain a resolution of the bands of alpha 1AT so as to be able to easily recognize all six PiM subtypes. The optimal resolution of the PiM subtypes is obtained without deforming the pattern of the Pi phenotypes. In addition to high resolution, ultrathin layer polyacrylamide gel isoelectric focusing permits a notable reduction of fees to 1/5 of the usual.

Adult↗