Layer Polyacrylamide Gel Research Articles

'Gel-Stacks' gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study.

Large DNA molecules (>20kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.

In Situ Single‐Cell Western Blot on Adherent Cell Culture

AbstractIntegrating 2D culture of adherent mammalian cells with single‐cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single‐cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin‐functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one‐step copolymerization process that creates protein‐decorated microwells. After single‐cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell‐based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab‐gel western blot, while revealing cell‐to‐cell heterogeneity in stress responses.

In Situ Single-Cell Western Blot on Adherent Cell Culture.

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

On-line microchip electrophoresis-mediated preconcentration of cationic compounds utilizing cationic polyacrylamide gels fabricated by in situ photopolymerization.

A simple and efficient method was developed for the fabrication of a cationic sample preconcentrator on a channel of a commercial poly(methyl methacrylate) (PMMA) microchip. This approach is based on a simple photochemical copolymerization for the fabrication of a permselective preconcentrator. The intersection of the PMMA microchip was filled with a gel solution comprising acrylamide, N,N-methylene-bis-acrylamide, (3-acrylamidopropyl) trimethylammonium, and riboflavin that functioned as a photocatalytic initiator. In situ polymerization near the cross of the sample outlet channel was performed by pinpoint irradiation with a 488 nm SHG laser beam, which is also used as the light source for fluorimetric detection. The electrokinetic property and electric repulsion between sample components and cationic groups on the polyacrylamide gel layer enables trapping and preconcentration of cations at the boundary of the anodic side of the gel layer. Reproducibility is about 20% RSD due to the variation of the position of the hand-made electrode in sample reservoir, but the preconcentration factors exceeded over 104-fold. The utility of the cationic preconcentrator gel was demonstrated by analyzing rhodamine derivatives, oligosaccharides labeled with rhodamine 110 and cytochrome C labeled with fluorescein isothiocyanate.

Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls.

During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC-MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.

Quantitative analysis of genomic element interactions by molecular colony technique

Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde–cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active β-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA–protein complex cannot be stabilized by formaldehyde cross-linking.

Mapping Three-Dimensional Stress and Strain Fields within a Soft Hydrogel Using a Fluorescence Microscope

Three-dimensional cell culture is becoming mainstream as it is recognized that many animal cell types require the biophysical and biochemical cues within the extracellular matrices to perform truly physiologically realistic functions. However, tools for characterizing cellular mechanical environment are largely limited to cell culture plated on a two-dimensional substrate. We present a three-dimensional traction microscopy that is capable of mapping three-dimensional stress and strain within a soft and transparent extracellular matrix using a fluorescence microscope and a simple forward data analysis algorithm. We validated this technique by mapping the strain and stress field within the bulk of a thin polyacrylamide gel layer indented by a millimeter-size glass ball, together with a finite-element analysis. The experimentally measured stress and strain fields are in excellent agreements with results of the finite-element simulation. The unique contributions of the presented three-dimensional traction microscopy technique are: 1), the use of a fluorescence microscope in contrast with the confocal microscope that is required for the current three-dimensional traction microscopes in the literature; 2), the determination of the pressure field of an incompressible gel from strains; and 3), the simple forward-data-analysis algorithm. Future application of this technique for mapping animal cell traction in three-dimensional nonlinear biological gels is discussed.

Transglutaminase-Induced Chemical and Rheological Properties of Cheese

Ca2+-independent transglutaminase isolated from Streptoverticillium mobaraense was used to obtain a novel cheese we called “crosslinked cheese.” We characterized both the cheese obtained by adding transglutaminase and milk clotting enzyme at the same time, and the one produced by adding transglutaminase, following cutting of the coagulum. In both cases, water content and cheese yields were higher, whereas protein content significantly increased from 33.8% to 39.5% only in the second type of crosslinked cheese. Isoelectric focusing of the protein extracted from the prepared cheese samples on a thin layer of polyacrylamide gel indicated that mainly β-caseins were involved in the transglutaminase-catalyzed crosslinks occurring in the curd. Few αs1-casein isoforms were entrapped in the curd matrix only when transglutaminase was added at the same time as the coagulant enzyme. Soluble nitrogen determination and HPLC peptide analysis indicated a markedly reduced proteolysis of crosslinked cheese during ripening, probably responsible for the observed enhancement of their hardness in comparison with the control cheese, obtained in the absence of transglutaminase.

In Situ Fabrication of Ionic Polyacrylamide-Based Preconcentrator on a Simple Poly(methyl methacrylate) Microfluidic Chip for Capillary Electrophoresis of Anionic Compounds

A simple and efficient method was developed for fabrication of an anionic sample preconcentrator on a channel of a commercial poly(methyl methacrylate) (PMMA)-made microchip using no photolithography or etching technique. The originality of our preconcentrator is based on simple photochemical copolymerization of monomers using the following procedure: All channels of the PMMA-made microchip were filled with gel solution comprising acrylamide, N,N'-methylene-bisacrylamide, and 2-acrylamide-2-methylpropanesulfonic acid with riboflavin as a photocatalytic initiator. In situ polymerization near the cross of the sample outlet channel was performed by irradiation with an argon ion laser beam, which is also used as the light source for fluorometric detection. The electrokinetic property and electric repulsion between sample components and anionic groups on the polyacrylamide gel layer produce, trap, and concentrate anions within a few minutes at the interface of the cathodic side of the gel layer. This method displays concentration factors as high as 10 (5). The availability of ionic preconcentrator was demonstrated by applying sensitive analysis of oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonate and some glycoproteins labeled with fluorescein isothiocyanate under various buffer systems.

Two-dimensional determination of dissolved iron and sulfur species in marine sediment pore-waters by thin-film based imaging. Thau lagoon (France)

A device composed of a polyacrylamide gel thin-film (18 cm high, 5 cm wide and 0.4 mm thick) and a PVC (polyvinyl chloride) film was used as a sediment probe to obtain iron and S(−II) sulfur dissolved species' distributions in sediment pore waters. A porous protective membrane was set on top of hydrogel layer. Probes were deployed in May 2003 for 24–48 h in the superficial sediment of Thau lagoon (France), in a shellfish farming area. The polyacrylamide gel layer was used as a DET (Diffusive Equilibration in Thin-films) device for 2D Fe(II) concentration determination, and as the diffusive layer of a DGT-like (Diffusive Gradients in Thin-films) device for sulfur species study. The accumulation layer of the DGT device consisted in a PVC film layer underneath the polyacrylamide layer. Iron determinations were performed by colorimetric methods with Ferrozine and imaging technique. Image acquisitions were performed with a flatbed scanner. Fe(II) concentrations were deducted from densitometry analysis of the magenta zones (ImageJ software). The calibration curve was obtained by densitometry analysis of polyacrylamide gel pieces which were equilibrated in known iron (II) concentration solutions. ΣFe distribution was performed but not quantified. Analysis of gray zones on the PVC layer provided a qualitative distribution of unidentified S(−II) dissolved compounds, related to H 2S zone for which information is obtained by classical methods (peeper and colorimetric measurements). EDX (X-ray energy dispersive spectrometry) and GIXR (Grazing-Incidence X-ray Diffraction) analyses of gray zone of this PVC layer provide evidence for FeS 2 catalyzed precipitation onto this film. Dissolved Fe(II) was mainly located near the sediment–water interface (SWI), showing a nonhomogeneous layer about 10 mm thick. Small Fe rich domains appeared deeper in the sediment and likely confirm newest paradigms in the field of sediment biogeochemistry. S(−II) species are detected from 3 to 4 cm below the SWI, with a heterogeneous spatial distribution showing a burrow-like structure.

Soybean seed pectinesterase

AbstractMethanol accumulates in axis tissues of maturing soybean seeds, correlating with preharvest seed deterioration. Accumulation of methanol appears to be associated with the enzymic demethylation of pectin methyl esters by pectinesterase (PE; EC 3.1.1.11). To characterize PE in developing and maturing soybean (Glycine max (L.) Merrill) seeds, enzyme activity was assayed in axis and cotyledon tissues. Activity per g fresh weight was 20–25 times higher in axes than in cotyledons with highest activities between 45 and 60 days after flowering (DAF). Twenty to 33% of the total PE activity was in the ‘soluble’ form (extracted with water, 0.5 M sucrose, 1 M sucrose and water). Soluble and cell-wall-bound PE (subsequently extracted with 1 M NaCI) were purified and characterized from axes of seeds at 45–60 DAF. Purification of PE was achieved through concentration of extracts by ultra-filtration, precipitation with ammonium sulfate (30–80% saturation), dialysis, gel filtration on Sephadex G-75 columns, and ion exchange chromatography on CM Sepharose CL-6B. Further purification of both soluble and bound PE was by isoelectric focusing (IEF) on ultrathin layers of polyacrylamide gel with simultaneous detection of protein and PE activity. It was possible to follow seven bands exhibiting PE activity with pl values between 6.0 and 9.5 in 1 M NaCI-extracts of total homogenates. Differences in the IEF patterns of bound and soluble PE were observed. Whereas the bound enzyme exhibited more basic PE bands (pl 8–9.5), the soluble enzyme had more active bands at pl 6.5, 7.0 and 7.5. The Mr was close to 33 000 and the pH optimum was 7.8 for both soluble and bound PE.

In situ measurement of dissolved phosphorus in natural waters using DGT

Due to the dynamic interactions of phosphorous species in natural systems, concentrations of individual species may change when samples are stored. Accurate analysis is, therefore, difficult unless measurements are made in situ. The technique of diffusive gradients in thin-films (DGT) has been developed for the in situ measurement of reactive phosphorus species in natural waters, sediments and potentially soils. Phosphorus species diffuse through a layer of polyacrylamide gel and then bind to ferrihydrite embedded in a further layer of gel. The mass of phosphorus in the ferrihydrite after a known deployment time is measured colorimetrically after acid elution. Concentration in the solution is then calculated using Fick's first law of diffusion. The diffusion coefficient of orthophosphate in the gel was independently measured to be 6.05×10 −6 cm 2 s −1 at 25°C which is 71% of the value in water. Using this value of the diffusion coefficient, DGT could be used to measure phosphate in solutions accurately without calibration. The preparation of the ferrihydrite is critical as irreproducible results were obtained when alternative iron oxide preparations were used. DGT measurements of reactive phosphorus species in situ in a eutrophic pond agreed well with mean replicate filterable reactive phosphorus (FRP) measurements made on a time series of collected water samples. For deployments of 24 h and 1 week, respectively, the limits of detection should be 0.07 μg l −1 of P and 0.01 μg l −1 of P. DGT was used to measure reactive phosphorus species in pore waters of a freshwater sediment at a spatial resolution of 1 mm.

Response of a thickness-shear mode quartz crystal resonator upon hydrogel formation

The response of an AT-cut quartz crystal piezoelectric resonator operating in the thickness shear mode (TSM) during polymer gel formation has been examined by acoustic network analysis. The series and parallel resonant frequencies as well as the amplitude and phase of the impedance of the TSM resonator were monitored in real-time when a layer of polyacrylamide gel was formed on one side of the sensor. Various concentrations of radical initiator and crosslinking agents were used to control the rates of polymerization and crosslinking during the hydrogel formation. The increase in the magnitude of the minimum impedance and the decrease in the maximum phase angle were observed as a result of the damping of both the magnitude-frequency and phase-frequency curves, caused by the transformation of the contacting medium from a viscous liquid to a viscoelastic hydrogel. Significant difference was observed between the responses of series and parallel resonant frequencies. The series resonant frequency increases while the parallel resonant frequency decreases during gel formation, and the magnitude of change in the parallel frequency is several times that in the series frequency. Small variations in the gelforming solutions such as the depletion of buffer and addition of double-stranded DNA cause distinct changes in the response profiles of the quartz resonator.

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.

The use of nuclear muprobe techniques to study the chemistry of lacustrine sediments and particles

The Oxford SPM has been used in two novel studies of lake chemistry: (a) The distribution of dissolved iron in sediment pore waters close to the sediment/water interface has been measured using the novel technique of diffusive equilibration in a thin film (DET). In this technique, which has a spatial resolution of < 1 mm, much less than that of competing techniques (1 cm), a thin layer of polyacrylamide gel is inserted into the sediment and after the rapid equilibration with the pore water, the gel is dried and fixed. The distribution of trace elements can then be measured using mubeam PIXE. Preliminary results have shown for the first time a subsurface maximum of Fe consistent with current theories of Fe dynamics. This paper presents some results obtained using the technique and discusses the limits on resolution and sensitivity, (b) Individual suspended lake particles (predominantly iron oxides and sulphides) have been analysed using point mubeam RBS and PIXE. Of particular interest in this study is the oxidation state of iron rich particles, so RBS with a 1 μm beam was used to determine the Fe: O stoichiometry of single particles. The particles were filtered from a depth of 14 m in Esthwaite Water in the English Lake District and handled in anoxic conditions until evacuation in the SPM sample chamber. Two distinct compositions of iron oxide were determined in clusters of about 5 μm diameter. Analysis by PIXE revealed that FeS was uniformly distributed in the particulate material and that it also contained elevated levels of Cu and Zn. This study was the first to demonstrate directly that discrete clusters of iron oxides are present in black particulate material which is commonly considered to comprise iron sulphides.

The zymogram method for detection of ribonucleases after isoelectric focusing: Analysis of multiple forms of human, bovine, and microbial enzymes

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 m permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T 1 and T 2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.

A method for DNA sequencing by hybridization with oligonucleotide matrix

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.

Electroblotting of Immobiline DryPlates applied to identification of human plasma apolipoprotein A-I.

A method for efficient electroblotting of Immobiline DryPlates, allowing subsequent immunological identification of separated proteins has been developed. A thin layer of 1% agarose containing sodium dodecyl sulfate is moulded on the 0.5 mm thick polyacrylamide gel surface after completed electrophoresis. After separation of the agarose-polyacrylamide gel sandwich from the plastic film the rigid gel sandwich could be easily transferred to a nitrocellulose membrane and electroblotting could be performed without adherence of the sticky polyacrylamide gel layer to the membrane. Using this technique human plasma high density apolipoprotein A-I isoforms, over a wide concentration range, could be identified in a heterogeneous mixture, conserving the isoform pattern and band sharpness produced in the immobilized pH gradient experiments.

Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 m urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.