Agarose Gel Beads Research Articles

Preparation of Low Cost Affinity Chromatographic Matrix and Its Application in Purification of a Lectin Isolated from Mulberry Seeds

Objective: Uniform sized agarose gel beads were prepared from agarose by emulsification technique. The prepared matrix has been attached with commercially available concanavalin A (conA) and finally it has applied to purify mulberry seed lectin. The present work shows a simple and inexpensive method for the preparation of an affinity matrix for purification of conA specific mulberry seed lectin. Method: Firstly, conA was immobilized on agarose gel beads and coupled to hexadiamine by using amino reactive bifunctional crosslinker (2,4,6-trichloro-1,3,5-trizine). Then it used as affinity matrix for the purification of mulberry seed lectin (designated as MSL). In purification protocol initially conA has been immobilized with agarose. Then MSL have been purified from the seeds of Morus alba L. Result: The agarose beads showed the best uniformity when 2-2.5% concentration of Tween 80 was used. The newly prepared affinity matrix agarose gel was able to purify MSL with the molecular weight of 22 kDa in a single step. The purified lectin strongly agglutinated with mice, chicken, bovine and human blood types A, B and O erythrocytes respectively. There was no activity found on goat erythrocytes. Conclusion: The present work shows a simple and inexpensive method for the preparation of an affinity chromatographic matrix. The prepared matrix was applied to purify mulberry seed lectin.

Development of an Ovine Model of Dilated Cardiomyopathy to Facilitate Clinically Translational Research

Preclinical studies of heart failure are critical in understanding disease pathophysiology and for development of novel therapies. Existing models that induce ventricular dysfunction suffer from various limitations, including their reversibility, minimal ventricular remodelling, or their highly invasive approach. Therefore, the aim of this research was to develop a percutaneous ovine survival model of cardiomyopathy that can be used for evaluation and optimisation of mechanical cardiac support and stem-cell therapies. Myocardial infarction (MI) was induced percutaneously using agarose gel bead solution to occlude obtuse marginal branches of the left circumflex coronary artery in merino sheep (n = 2). Comparatively, mitral regurgitation (MR) was induced (n = 2) by disrupting the chordae tendinae of the posterior leaflet of the mitral valve. Serial echocardiographic and myocardial strain measurements were performed pre- and post–procedure and weekly for 16 weeks. Electrocardiographic ST-segment elevation post-MI correlated with echocardiographic changes. Mean reduction in left ventricular ejection fraction immediately post–procedure was 18.8% and reduction in circumferential and radial strain was seen secondary to regional wall motion akinesis. Ejection fraction was reduced from 77% to 47% over 16 weeks. End-diastolic volume increased in the MI sheep by 92% and the MR sheep by 84%. We have demonstrated the feasibility of a percutaneous ovine infarct and MR survival model in producing progressive LV dilation and systolic dysfunction. Ultimately, the development of this clinically relevant survival model of heart failure will be critical for the evaluation of novel therapies.

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 μM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.

Fabrication of bimodal-pore SrTiO3 microspheres with excellent photocatalytic performance for Cr(VI) reduction under simulated sunlight

Solving the increasing contamination from toxic heavy metal ions in wastewater is an imperative issue in photocatalysis research area. In this study, three-dimensional (3D) porous SrTiO3 microspheres have been fabricated by a sol–gel-templating method using the agarose gel bead containing SrCO3 granules as the template. The resultant SrTiO3 microspheres, several tens of micrometers in diameter, exhibit a bimodal pore structure, in which the macropore about 70–150nm in size stems from SrCO3 granules and the mesopore about 3nm is formed via removing the agarose fiber embedded in the composite microspheres. The porous framework of SrTiO3 microspheres is assembled by regular single-crystalline SrTiO3 nanocubes with an edge length of 100±10nm. The highly interconnected porous network renders numerous pathways for the rapid mass transport, strong adsorption of reactants and multi-reflection of incident light. Moreover, the as-prepared SrTiO3 microspheres exhibit excellent photocatalytic performance for the Cr(VI) reduction under simulated sunlight, which can reduce nearly 100% Cr(VI) at pH 2 within 2h and retain a relatively high reduction ability after six recycles.

Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

Tuning the Mechanical Properties of Hydrogel Core-Shell Particles by Inwards Interweaving Self-Assembly.

Mechanical properties of hydrogel particles are of importance for their interactions with cells or tissue, apart from their relevance to other applications. While so far the majority of works aiming at tuning particle mechanics relied on chemical cross-linking, we report a novel approach using inwards interweaving self-assembly of poly(allylamine) (PA) and poly(styrenesulfonic acid) (PSSA) on agarose gel beads. Using this technique, shell thicknesses up to tens of micrometers can be achieved from single-polymer incubations and accurately controlled by varying the polymer concentration or incubation period. We quantified the changes in mechanical properties of hydrogel core-shell particles. The effective elastic modulus of core-shell particles was determined from force spectroscopy measurements using the colloidal probe-AFM (CP-AFM) technique. By varying the shell thickness between 10 and 24 μm, the elastic modulus of particles can be tuned in the range of 10-190 kPa and further increased by increasing the layer number. Through fluorescence quantitative measurements, the polymeric shell density was found to increase together with shell thickness and layer number, hence establishing a positive correlation between elastic modulus and shell density of core-shell particles. This is a valuable method for constructing multidensity or single-density shells of tunable thickness and is particularly important in mechanobiology as studies have reported enhanced cellular uptake of particles in the low-kilopascal range (<140 kPa). We anticipate that our results will provide the first steps toward the rational design of core-shell particles for the separation of biomolecules or systemic study of stiffness-dependent cellular uptake.

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

BackgroundSeveral recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.ResultsWe describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.ConclusionTM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1236-7) contains supplementary material, which is available to authorized users.

Preparation of affinity adsorbents and purification of lectins from natural sources.

Lectins are purified by affinity chromatography to take advantage of their carbohydrate-specific interactions. Highly efficient affinity adsorbents are powerful tools to obtain homogeneous lectins with distinct specificities. Here, we describe three methods to prepare affinity adsorbents by immobilizing carbohydrates or glycoconjugates on agarose gel beads. Because the ligands are immobilized via a stable and nonionic linkage under mild conditions, the adsorbents possess high binding capacity for lectins with low nonspecific adsorption and can withstand repeated use. The procedures require neither specialized techniques and apparatus nor highly toxic compounds. Using these adsorbents, many plant and animal lectins can be purified in a few steps.

Improved method for coupling of DNase I to a crosslinked agarose gel bead

Improved method for coupling of DNase I to a crosslinked agarose gel bead

Development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody

Two kinds of monoclonal antibodies (MoAbs), OCA-10A and OCA-1B, were prepared based on their specificity to ochratoxin A (OTA) and ochratoxin B (OTB) and on their tolerance to 40% methanol. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC50) value of OCA-10A was 27ng/mL for OTA and 17ng/mL for OTB, and that of OCA-1B was 28ng/mL for OTA and 13ng/mL for OTB. Immuno-affinity columns (IACs) using these MoAbs were prepared with agarose gel beads. The IAC with OCA-1B showed a NaCl-dependent binding ability to OTA and OTB, while interestingly, the IAC with OCA-10A bound to them without NaCl. The IAC with OCA-10A showed a high methanol tolerance when compared with existing IACs, as expected from the high methanol tolerance of OCA-10A itself. Such tolerance was maintained for the application of the cocoa extract with 70% methanol and the wheat extract with 60% acetonitrile, while the tolerance was slightly altered by interference from the cocoa extract. Examinations with organic solvents at higher concentrations than the allowable level in existing IACs showed that OTA and OTB spiked with wheat, cocoa and red wine could be purified with high recovery. The newly developed IAC is expected to show sufficient clean-up ability for food analyses.

Metal/semiconductor separation of single‐wall carbon nanotubes by selective adsorption and desorption for agarose gel

AbstractMetallic and semiconducting single‐wall carbon nanotubes (SWCNTs) were separated by selective adsorption and desorption using agarose gel beads and SWCNTs/sodium dodecyl sulfate dispersion. Comparison to the batch method, the purities of metallic and semiconducting SWCNTs obtained by a column method were improved to 90 and 95%, respectively. When linear‐gradient elution was applied to the column method, semiconducting SWCNTs and presumed mixed bundles could be separated into early‐ and late‐eluted fractions, respectively. In the semiconducting fractions, chirality separation was also detected.

Development of a Novel Immunoaffinity Column for Aflatoxin Analysis Using an Organic Solvent-Tolerant Monoclonal Antibody

An organic solvent-tolerant monoclonal antibody specific to aflatoxins B(1), B(2), G(1), G(2), and M(1) (AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1)) was prepared. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) values were 1.9, 2.1, 2.1, 2.4, and 2.8 ng/mL for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1), respectively. Antibody reactivity was retained at 40% methanol concentration or at acetonitrile concentrations up to 40%. An immunoaffinity column (IAC) was prepared using agarose gel beads with bound antibody. The IAC retained the tested AFs that were 89, 90, 95, 90, and 89% for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1) at 20% acetonitrile concentrations or that were 81, 87, 79, and 83% for AFB(1), AFB(2), AFG(1), and AFG(2) at 60% methanol concentrations. Roasted peanuts and seven kinds of spices were spiked with 8.0, 1.0, 6.0, and 1.0 ng for AFB(1), AFB(2), AFG(1), and AFG(2) per 1 g sample and extracted with 90% acetonitrile. The roasted peanuts and cayenne pepper out of the spices were also extracted with 70% methanol. The extracts were diluted 5-fold with phosphate-buffered saline and applied to the IAC. The spiked aflatoxins were recovered with satisfactory rates: 78 (RSD, 2.1%) to 127% (RSD, 1.7%). The developed IAC was used for the analysis of aflatoxins in naturally contaminated samples of roasted peanuts and cayenne pepper. The newly developed IAC showed substantially organic solvent tolerance at the concentration that could not be used for existing IACs, and the column showed good ability to clean up samples for food analysis.

Characterization and biological application of YAG:Ce3+ nanophosphor modified with mercaptopropyl trimethoxy silane

We focus on the inorganic nanophosphor of YAG:Ce3+ nanoparticles as an alternative fluorescent probe instead of organic dyes for biological application. YAG:Ce3+ nanoparticles have the green emission at 530 nm under the excitation of blue light at 450 nm. Conventional biochemical equipments for organic dyes can be used for YAG:Ce3+ nanoparticles. SH groups were introduced by surface modification of YAG:Ce3+ nanoparticles with 3-mercaptopropyl trimethoxy silane, and were characterized by X-ray fluorescence analysis, FT-IR, and Ellman method. We demonstrated tagging avidin-immobilized agarose gel beads with biotinylated-YAG:Ce3+ nanoparticles, and tagging rabbit IgG-immobilized agarose gel beads with antirabbit IgG-immobilized YAG:Ce3+ nanoparticles.

Design of a Specific Peptide Tag that Affords Covalent and Site-Specific Enzyme Immobilization Catalyzed by Microbial Transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments.

Arc-synthesized single-walled carbon nanotubes have been purified through preparative electrophoresis in agarose gel and glass bead matrixes. Two major impurities were isolated: fluorescent carbon and short tubular carbon. Analysis of these two classes of impurities was done. The methods described may be readily extended to the separation of other water-soluble nanoparticles. The separated fluorescent carbon and short tubule carbon species promise to be interesting nanomaterials in their own right.

Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme‐linked immunosorbent assay system

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.

Changes induced by DC electrical field in agar, agarose, alginate and gellan gel beads

Gel beads produced from agar, agarose, alginate and gellan were interposed between a pair of electrodes in a special custom-made apparatus in order to study weight, mechanical and structural changes within them. Their shrinkage was regularly accompanied by electrolysis. The degree of weight loss depended on time and intensity of the DC electrical field, compositions of the bead and the solution in which it was immersed. At a field strength of 64 V/cm, weight losses of ∼22, 24, 35 and 47% of initial weight were observed for agarose, alginate, gellan and agar, respectively. The rate at which the reduction in weight loss occurred was also dependent on the osmotic pressure outside the bead. Contraction took place only in the region of the anode, and was associated with the electrohydrodynamic transport of hydrated ions and concomitant water exudation. The affected area of the bead resembled the shape of the electrode. The spheroid shape of the gel beads did not change significantly after DC contraction. Agarose appeared to be the least affected, with no effect on the inner layers. Agar beads revealed affected inner layers, and gellan and alginate beads were even more influenced. The beads' distortion towards a flatter shape could be beneficial in terms of their spatial arrangement, as well as more convenient for, example, insertion under the skin for transplantation and slow release of medicinal agents.

Influence of the enzyme derivative preparation and substrate structure on the enantioselectivity of penicillin G acylase

The immobilization of an enzyme on solid support can give a catalyst with improved stability and catalytic properties compared to the native enzyme; in this work the enantioselectivity of penicillin G acylase (PGA - EC 3.5.1.11) isolated from different microbial sources ( Escherichia coli, Kluyvera citrophila and Arthrobacter viscosus) has been studied. Two matrixes (Eupergit C and agarose gel beads) and different binding techniques have been investigated measuring the enantioselectivity of the different immobilized enzyme in the hydrolytic resolution of racemic esters and amides of mandelic acid. We have found a relevant dependence of the catalytic properties on the enzymatic source, immobilization technique and substrate structure. The best result has been obtained using the acylase from E. coli immobilized on glyoxyl agarose by multipoint interaction in the hydrolysis of mandelic acid iso-propylamide at pH 6.5 and 4 °C (73% enantiomeric excess at 47% of conversion). These results suggest that the immobilization of the PGA from E. coli on different supports (via different orientations, with different rigidity) may be a useful tool to modulate the catalytic properties.

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.