Cross-linked Polyacrylamide Gels Research Articles

Study by response surface methodology of the swelling kinetic of weakly basic polyacrylamide gels in water

Abstract The aqueous swelling kinetic and cooperative diffusion coefficient were studied for various crosslinked polyacrylamide gels, copolymerized with N,N-dimethylaminoethyl methacrylate (DMA), containing different crosslinker ratios, monomer concentrations and percentages of ionizable monomer. The cooperative diffusion coefficient was determined using a theory developed by Li and Tanaka for the swelling kinetics of disk gels. Using experimental design, response surfaces were calculated for the cooperative diffusion coefficient as a function of gel composition. The results obtained show that the degree of crosslinking and the percentage of ionizable monomer are the variables which most strongly affect the cooperative diffusion coefficient.

Immobilization and characterization of beta-galactosidase from the plant gram chicken bean (Cicer arietinum). Evolution of its enzymatic actions in the hydrolysis of lactose.

beta-Galactosidase (beta-D-galactosidase galactohydrolase, EC 3.2.1. 23) isolated and purified from gram chicken bean was immobilized on cross-linked polyacrylamide gel. The activity yield was high and attained up to 72%. Compared with the free enzyme, the immobilized enzyme had a wider operational pH range and better thermal stability. Lyophilized pieces exhibited good stability when stored at room temperature for 60 days and a favorable operational stability when used eight times repeatedly without loss of enzymatic activity under the same conditions. Kinetic data (K(m), V(m), and E(a)) for the free and the immobilized enzymes were determined using O-nitrophenyl-beta-D-galactoside (ONPG) and lactose as substrates. The result of time courses of hydrolysis of lactose showed that beta-galactosidase from the plant gram chicken bean would have a promising application in the hydrolysis of lactose in milk.

高速液体クロマトグラフィー（ＨＰＬＣ）によるスチレン（Ｓｔ）‐メタクリル酸メチル（ＭＭＡ）‐アクリロニトリル（ＡＮ）三元共重合体の組成分布解析

Separation of styrene-methyl methacrylate-acrylonitrile terpolymers by chemical composition was studied using high-performance liquid chromatography(HPLC). Cross-linked polyacrylonitrile gel or polyacrylamide gel as a stationary phase and mixture of chloroform, acetonitrile and n-hexane as an eluent were used in the normal phase HPLC. For the reversed-phase HPLC, polystyrene gel and a mixture of chloroform and acetonitrile were used as a stationary phase and an eluent, respectively. Solvent gradient of increasing chloroform content was utilized. In the case of normal phase HPLC terpolymer was eluted from the non-polar polymer, while from the polar polymer in the case of reversed-phase HPLC. Individual terpolymers were completely separated from a mixture of nine terpolymers by cross-fractionation using the combination of HPLC with acrylonitrile and acrylamide column or acrylonitrile and styrene column system.

Templated Pores in Hydrogels for Improved Size Selectivity in Gel Permeation Chromatography

The pore structures of cross-linked polyacrylamide gels can be altered by polymerizing in the presence of high concentrations of unreactive, micellar surfactant cosolutes which act as "templates". Removal of surfactant after polymerization is expected to leave pores with the approximate shape and dimensions of the surfactant micelles. A simple model was developed to simulate gel permeation chromatography (GPC) separations of globular proteins on templated gels. The model assumes that the partition coefficient for sieving of a protein is equal to the fraction of gel volume accessible to a sphere with a radius equal to the protein Stokes radius. The total gel volume is considered to include a fraction that is a conventional, random gel matrix and a remaining fraction contributed by templated pores. The pore size distribution of the conventional gel was estimated using the Ogston equation, which approximates the matrix as a random collection of long, thin, rigid fibers. Templated pores were assumed to have a Gaussian distribution of radii centered about some mean determined by the micelle radius. In comparison to conventional media, gels with templated pores are predicted to exhibit more sharply defined exclusion limits and improved resolution over a narrow size range centered on the mean templated pore size. Selectivity and resolution are expected to increase as the volume fraction of templated pores is increased and as the dispersion of templated pore radius is decreased. Small changes in template radius lead to large changes in the molecular weight range of optimal separation of globular proteins. It should be possible to create a series of GPC media that collectively offer high resolution over the molecular weight range of most globular proteins of interest.

Effects of including dry polyacrylamide gel with seed on establishment of alfalfa and Russian wildrye

To examine the possible benefits of crosslinked polyacrylamide gel when establishing forages in a semiarid environment, applications of dry gel in fine, medium and coarse granular forms were made at rates from 0.25 to 4.00 g m−1 of row with seed of alfalfa (Medicago sativa L.) and Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] in each year 1992–1994 at Swift Current, Saskatchewan. The gel had no effect on seedling year establishment or first year forage production on a sandy-loam soil. On a nearby sandy-clay loam soil, in one of 3 yr with alfalfa, and 2 of 3 yr with Russian wildrye, the lowest gel rate decreased establishment compared with the check, with a further reduction as the rate of gel increased. Alfalfa forage yields were not affected. Gel application decreased Russian wildrye forage yield in 1 of 3 yr. Key words: Medicago sativa, Psathyrostachys juncea, forage yield, soil texture

A three-dimensional phase boundary model for diffusion processes involved in reactions of crosslinked polymeric amines with low molecular weight esters

The reactivity of amino groups attached to N-N′-methylene-bis-acrylamide (NNMBA)-and triethyleneglycol dimethacrylate (TEGDMA)-crosslinked polyacrylamide gels (2–20 mol% crosslinked) has been investigated by following the aminolysis ofp-nitrophenyl ester of benzoyl glycine in a mixed solvent. A differential method was employed for following the rate of aminolysis reaction at definite intervals of time. The extent of aminolysis was found to be maximum between 25 and 35 min after initiation for these resins. The rate constants were calculated using the equation for three-dimensional phase boundary model with spherical symmetry and three-dimensional diffusion model. The reaction appears to be a phase boundary process up to 40 min after initiation and thereafter is fully controlled by three-dimensional diffusion. The rate constants do not show any dispersion when this model is used.

Gel electrophoresis of DNA fragments in narrow-bore capillaries.

In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2-10 microm, filled with a cross-linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser-induced fluorescence (LIF)-detection was employed utilizing a 488 nm argon ion laser for separations of C- and T-terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 microm to 10 microm resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 microm ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.

Application of capillary gel electrophoresis to the diagnosis of the aldehyde dehydrogenase 2 genotype.

This study dealt with the application of capillary gel electrophoresis (CGE) to diagnosis of the aldehyde dehydrogenase 2 (ALDH-2) genotype. Electrophoresis was performed on a low cross-linked polyacrylamide gel ¿3% T [g acrylamide+g Bis (N,N'-methylenebisacrylamide)], 0.5% C (g Bis/% T)¿ in 100 mM Tris-borate buffer (pH 8.3) at -10 kV with on-column UV detection (260 nm). During the PCR reaction, DNA from the wild-type allele generated a MboII restriction site, which is an amplification created restriction site. This did not occur, however, with DNA fragments from the mutant allele. Therefore, determination of the heterozygous genotype, the coexistence of wild-type and mutant alleles, was easily possible. Analysis of the MboII restriction digests of the PCR products was completed in less than 20 min, showing two peaks corresponding to fragments of 125 (cleaved) and 135 (uncleaved) base pairs (bp), respectively. On the other hand, determination of the homozygous genotype, wild-type or mutant, was difficult in one electrophoresis run. The CGE of the MboII restriction digests gave a single peak and the identification, cleaved or uncleaved, was difficult under our experimental conditions. However, the addition of aliquots of the PCR reaction mixture to the restriction digests, followed by re-electrophoresis, allowed successful diagnosis, yielding two peaks (cleaved and uncleaved) for the wild-type and one peak (uncleaved) for the mutant allele. This study demonstrated that CGE offers a high-speed, high-resolution analytical tool for determining genetic types, as compared with the conventional slab gel methodologies.

Oligomeric Separation of Ionic and Nonionic Ethoxylated Polymers by Capillary Gel Electrophoresis

Capillary gel electrophoresis (CGE) has proven itself as a superior, high-resolution technique for separating proteins, peptides, oligonucleotides, and other naturally occurring molecules. In the years since its inception, few applications of CGE to nonbiological synthetic polymers have been reported. CGE has been applied to the separation of ionic and nonionic ethoxylated surfactants and poly(ethylene glycol) (PEG) oligomers. Oligomer distributions of several sulfated and phosphated alkylphenol ethoxylate surfactants have been baseline resolved with CGE on commercial cross-linked polyacrylamide gel columns. Nonionic surfactants and PEG oligomers were derivatized with phthalic anhydride in order to provide charge and detectability. PEG oligomers ranging from ethylene glycol to species containing more than 120 ethylene oxide units have been resolved. A linear relationship between migration time and molecular weight was found, which indicates that the separation mechanism is not simply based on molecular size but is also influenced by the electrophoretic mobility of the oligomers. The main drawbacks of CGE include relatively long analysis times and somewhat fragile and expensive columns.

Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels

The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30.

Nonradioactive multiplex PCR screening strategy for the simultaneous detection of multiple low-density lipoprotein receptor gene mutations.

We have developed a rapid, nonradioactive screening test enabling the simultaneous analysis of three low-density lipoprotein receptor (LDLR) gene mutations (D154N, D206E, and V408M), which together account for familial hypercholesterolemia (FH) in approximately 90% of the South African Afrikaner population. The assay is designed so that FH patients, negative for these founder-related mutations (found in descendants of European settlers), subsequently can be screened for unknown mutations in the mutation-rich exon 4 of the LDLR gene. Our screening assay consists of two steps: (1) multiplex allele-specific PCR amplification of exons 4 and 9, and (2) simultaneous analysis of single- and double-strand conformational polymorphisms in exon 4 by vertical electrophoresis on low cross-linked polyacrylamide gels. The simplicity, specificity, and versatility of the multiplex assay makes it an ideal system for routine screening of FH mutations in large population samples.

Sodium dodecyl sulfate-gel electrophoresis of proteins employing short capillaries.

The performance of capillary gel-sodium dodecyl sulfate (SDS) electrophoresis for molecular-mass-dependent separation of proteins has been examined employing cross-linked polyacrylamide gels and untreated fused-silica capillaries of short length. An apparatus for capillary electrophoresis has been constructed, combining a UV detector equipped with a capillary holder, a power supply, and a micromanipulator for sample loading. The apparatus is capable of accommodating gel capillaries of different lengths and can be operated with two methods of sample loading: manual injection on top of the gel or electrokinetic injection. Standard proteins have been separated according to their molecular mass (ranging from 17000 to 116000) within 30 min, employing gel capillaries of effective lengths shorter than 10 cm and polyacrylamide gels ranging from 2.4 to 4.8%T (5%C). The results confirmed that gel capillaries of effective length less than 10 cm can be used for protein size-separation by SDS electrophoresis, with much higher performance than is achieved in rod gel-SDS electrophoresis.

Studies of some experimental conditions for the polymerization of the continuous beds (cross-linked polyacrylamide gels) in order to improve their chromatographic properties

Continous chromatographic beds are synthesized by a simple and cost-effective procedure which does not involve the conventional, time-consuming preparation of beads. The aim of this study was to improve the flow properties of the beds by investigating how they are affected by polymerization temperature, the pH of the buffer in which the polymerization takes place, the nature of the constituents of this buffer, the concentrations of catalyst and accelerator and the concentration of the precipitating agent (ammonium sulfate). The infuence of flow rate and the time constants of the detector and recorder on peak width was also studied.

Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies

We have demonstrated that the gel shift assay, a powerful method to study protein · DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA · DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein · DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA·DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants ( K d) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.

Crosslinked poly(N‐isopropylacrylamide) gel for electrophoretic separation and recovery of substances

AbstractCrosslinked poly(N‐isopropylacrylamide) gel was tested on the feasibility for a preparative electrophoretic matrix. Horse heart myoglobin and bovine hemoglobin were well separated on the gel matrix electrophoretically by molecular sieving effect of the gel network. Relative mobilities of those proteins in the gel were larger than those in a crosslinked polyacrylamide gel of the same polymer concentration. After the separation, the protein‐containing portion of the gel underwent swelling at 4°C and deswelling at 37°C, alternatively. As a result of the deswelling, each protein was recovered in a discharged solution out of the gel at almost 100% yield.

Analysis of polymerase chain reaction product by capillary electrophoresis and its application to the detection of single base substitution in genes

Capillary gel electrophoresis (CGE) was studied for the direct analysis of polymerase chain reaction (PCR) amplified samples. A low cross-linked polyacrylamide gel (3%T, 0.5%C) was used for CGE with treated and untreated silica capillaries. CGE showed high reproducibility and resolution in the separation of DNA fragments ( ca. 100–1000 base pairs) produced by PCR. The CGE system was applied to the detection of an amplification refractory mutation system (ARMS) and PCR—restriction fragment length polymorphism (PCR-RFLP), which are detection methods of single base substitution in genes using PCR. With the CGE system, full automation of PCR product detection is feasible.

Preparation and characterization of a stable polyacrylamide sieving matrix-filled capillary for high-performance capillary electrophoresis

A stable sieving matrix of polyacrylamide filled in a capillary was developed. The inner wall of a fused-silica capillary was covalently bonded with a linear polyacrylamide through SiC linkages, in which cross-linked polyacrylamide gel or linear polyacrylamide solution was filled. The stability of the coating was examined by exposure of the capillaries to alkaline buffer (pH 8) for up to 30 days. Compared with the coatings with linear polyacrylamide bonded through siloxane linkages, the present capillary markedly reduced the electroosmotic flow. Thus, the sieving matrix in the capillary was stabilized, resulting in a prolonged lifetime of the capillary and good reproducibility of separations. The migration behaviours of oligonucleotides were compared for the cross-linked gel and linear polyacrylamide solution at the same concentration.

Manufacture of B-ZSM-5 structural type boralites by hydrothermal synthesis: F. Skoda; J. Bauer Vysoka Skola Chemicko Technologicka Czech 274,790, Nov. 12, 1991; Appl. Jan 18, 1990

A phenolic resin cross-linked nonionic polyacrylamide (NPAM) gel used for in-depth profile control was systematically analyzed. Long gelation time and strong gel strength of the gel system was obtained by adjusting NPAM or cross-linker concentration. An increase in temperature and salt concentration accelerated the gelation process and improved the gel strength. Although shearing brings a negative effect on gel performance, the gel system still had strong gel strength which could make it long-term stability in in-depth formation. Differential scanning calorimetry determined that this gel system should be used for oilfield application at temperatures below 143 °C. A compact three-dimensional network structure was formed in the bulk gel system and in porous media which contributed to long-term gel stability in the formation. Sand-pack flow experiments showed that the NPAM gel had a good plugging capacity and could selectively plug high permeability zones. By retention, adsorption, and bridging across the pore throats, the gel systems effectively reduced the permeability of porous media in high permeability zones and diverted fluid into low permeability zones, and thus improved the formation profile. Furthermore, this gel system for in-depth profile control treatment was successfully used in the Xinjiang oilfield of China which provided a reference for other similar high water cut oilfields for water control worldwide.

Characterization of over‐expressed alkaline phosphodiesterase I in tumour‐derived fibroblasts from patients with neurofibromatosis

Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6.0. The enzyme required Zn2+ for its activity, was heat labile, and nicked superhelical covalently closed circular phi X174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430,000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.