Agarose Concentration Research Articles

Responsiveness of individual airways to methacholine in adult rat lung explants.

We used a modified adult lung explant technique to directly measure the area of individual airways before and after methacholine (MCh) administration. Lungs were removed from 12-wk-old male Lewis rats under sterile conditions, filled with an agarose-containing solution at 37 degrees C, and cooled to 4 degrees C. Transverse slices (0.5-1.0 mm thick) were cut and cultured overnight. Concentration-response curves to MCh were determined for explant airways from lungs inflated to 25, 50, 75, and 100% total lung capacity (TLC) with a 1.0% agarose solution and to 75% TLC with 0.5 and 2.0% agarose solutions. MCh was added to the medium to achieve final concentrations ranging from 10(-9) to 10(-2) M. Airways were imaged before and 10 min after each increase in MCh concentration with an inverted microscope and video camera, and airway area was determined by computerized image processing. The maximal response (MR) ([1-(minimal area/baseline area)] x 100) and concentration of MCh resulting in 50% MR (EC50) were determined. A total of 217 airways from 3-12 explants per rat constricted in a concentration-dependent manner. Baseline area was larger with both higher lung volumes and agarose concentrations. MR was greatest in the airways from the 25% TLC and 0.5% agarose explants. Although there was considerable heterogeneity toward MCh within rats (EC50 varied up to 5.46 x 10(5)-fold), the median EC50 was similar among all rats (range 1.96 x 10(-6)-5.87 x 10(-4) M). Lung inflation volume and agarose concentration affected baseline area and MR, suggesting that airway-parenchymal interdependence mechanisms are operative in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks.

Size-Dependent Separation of Proteoglycans by Electrophoresis in Gels of Pure Agarose

An improved system for electrophoresis of proteoglycans is presented, using a discontinuous buffer system that allows stacking of the sample. The molecular sieving in pure agarose was studied using cartilage proteoglycans separated into fractions of different size (Kav value) by gel chromatography. These fractions were used to establish the relationship between size and mobility in agarose gels of different concentrations. With low agarose concentration, the separation was largely dependent on charge density. With increasing agarose concentration, the separation became increasingly dependent on size. Electrophoresis provides a suitably sensitive tool for analysis of the proteoglycan and glycosaminoglycan fractions prepared by the Alcian blue precipitation described in the preceding paper. Alcian blue precipitated cartilage proteoglycans appeared identical in size to molecules prepared by conventional procedures when analyzed by electrophoresis. Proteoglycans of various sizes were prepared by precipitation with Alcian blue of 4 M guanidine-HCl extracts of bovine nasal cartilage, human articular cartilage, human skin, and bovine sclera. Proteoglycans were also precipitated from human synovial fluid and plasma. The samples were electrophoresed on 1.2% agarose gels, which was the highest agarose concentration suitable for large aggregating proteoglycans but still separating the smallest proteoglycans with one or two glycosaminoglycan chains from each other. Several sizes of proteoglycans were found in each sample except blood plasma, which only contained one small proteoglycan/glycosaminoglycan. Electrophoresis in agarose is also suitable for separating proteoglycan aggregates and monomers and allows rapid analysis of several samples at a time. The proteoglycans prepared by Alcian blue precipitation retained their ability to form aggregates with hyaluronic acid. In addition to separation according to size, the electrophoresis method is also useful for separating free glycosaminoglycan chains according to charge. Electrophoresis in 0.6% agarose was used to demonstrate that both chondroitin sulfate and keratan sulfate were precipitated with Alcian blue at 0.4 M guanidine-HCl. No keratan sulfate, either alone or together with chondroitin sulfate, was precipitated at 1.0 M guanidine-HCl concentration.

Electrophoresis of Neurochordins, a Family of High Molecular Weight Neural Tissue Glycoproteins, on Horizontal Submerged Agarose Gels in the Presence of Dodecyl Sulfate

We have developed a procedure for the use of agarose gels to separate electrophoretically neurochordins, a family of high molecular weight neural tissue glycoproteins. This procedure is a modification of the method of horizontal submerged electrophoresis on agarose gels routinely applied for the separation of DNA restrictions. For protein analysis we prepared gels of higher agarose concentration (3%), and 0.1% of sodium dodecyl sulfate was present in the buffer used both for gel formation and as an electrode buffer. After electrophoresis agarose gels can be directly stained with Coomassie blue, or proteins can be transferred from the gel to nitrocellulose or nylon filters for immunostaining. The main advantage of the proposed method compared to the method of electrophoresis on large-pore, composite agarose/polyacrylamide gels is its simplicity. Other proteins of very high molecular weight can also be successfully separated by this technique.

The use of agarose gels for quantitative determination of fluid saturations in porous media

The use of agarose gel reference standards for quantifying petrophysical properties in porous media is described. The specific interest is to determine the values of fluid saturations and porosity in oil bearing rocks; the MRI methodology for estimating these properties is discussed. It is shown that the relaxation times of the gel reference standard and the relaxation times of the fluid contained in the porous media affect the estimation process. The determination of porosity and fluid saturations can be greatly simplified if the relaxation times of the reference standard and the relaxation times of the fluid are closely matched. Gel concentration of paramagnetic impurities in the form of copper ions is used to control the longitudinal relaxation properties. The relaxation properties of agarose gels, as a function of agarose and paramagnetic impurity concentrations, have been measured at 2.0 T. The data are well fitted by a simple polynomial in agarose concentration and paramagnetic impurity concentration. Finally, a one-dimensional imaging example of use of agarose gels as reference phantoms is discussed.

DNA digest analysis with capillary electrophoresis.

DNA digest analysis with polymer-filled capillaries in capillary electrophoresis is described. The samples analyzed consisted of commercially available standards including a 100 base pair ladder with repeating units up to 2000 base pairs. Three DNA digests covered the most common small fragment ranges: up to 600 base pairs, up to 1500 base pairs and from 100 to 2500 base pairs. All samples were separated by traditional slab gel electrophoresis at various agarose concentrations and by automated capillary electrophoresis. The capillary electrophoretic separations were achieved with noncross-linked polyacrylamide from 6% monomer solutions. The acrylamide was polymerized inside the capillary, which was coated with a methacryloxysilane to insure binding of the polyacrylamide to the capillary wall. With 6% columns, excellent separations were observed up to 600 base pairs with base line resolution for fragments differing in less than 10 base pairs. Resolution power for fragments between 600 and 2200 decreased to about 300 base pairs. Compared to slab gels, capillary electrophoresis achieved better resolution in the low fragment range, whereas with the reported column composition, slab gels were superior above 600 base pairs. Fast access to the analysis of a specific sample, automation, and a larger dynamic range for sample load are further benefits of a DNA digest analysis by capillary electrophoresis.

Investigation of the tissue equivalence of gells used for NMR dosimetry

The transition of Fe2+ to Fe3+ in Fricke solution after irradiation results in a change of NMR proton relaxation times in agarose gels which can be used for the dosimetry of ionizing radiation. The main advantage of this system is the possibility of observing dose distributions in 3 dimensions in a medium which is supposed to be tissue equivalent. The aim of the study was to quantify parameters which determine the tissue equivalence of NMR dosimetry gels. Electron densities and effective atomic numbers were calculated for gels with varying iron, sulphur and agarose concentration. The Hounsfield CT numbers so derived agree well with the CT numbers measured on a clinical CT scanner (effective photon energy 70.7 keV).

Ni-DTPA doped agarose gel—A phantom material for Gd-DTPA enhancement measurements

In order to study the relationship between the concentration of Gd-DTPA in tissue, and the resulting changes in relaxation times and signal intensity, a phantom material that has similar relaxation times to tissue and that can be doped with Gd-DTPA is required. The “tissue-equivalent” material should not contain Gd; nor should it alter the relaxivities of Gd-DTPA from their values in aqueous solution ( R 1 = 4.5 sec −1 mM −1; R 2 = 5.5 sec −1 mM −1 at 1.5 T). Conventional materials, based on CuSO 4-, MnCl 2-, or GdCl 3 LaCl 3 -agarose mixtures, are not suitable, since Gd is displaced from the Gd-DTPA chelate. The new material, consisting of Ni-DTPA dissolved in agarose, is easy to prepare and dose not interact with Gd-DTPA. Its relaxation times are stable; relaxivity R 1 was within 4% of its aqueous value over 109 days. T 1s have low dependence on temperature (0.2–1.0%/°C at 21°C) and on field strength, allowing the material to be used as a relaxation time standard for quality assurance. Equations giving the concentration of Ni-DTPA and agarose to produce a required T 1 and T 2 are provided.

Influence of the agarose matrix in pulsed-field electrophoresis.

Properties of agarose potentially relevant to PFGE (pulsed-field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100,000 to 200,000 Da, but this is not tightly related to the effective gel strength. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strength, non-borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda-phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.

Agarose for a bioartificial pancreas.

Islets were encapsulated into 5% concentration agarose microbeads. The effect of microencapsulation on islet allograft survivals was determined using a streptozotocin-induced diabetic (STZ) mouse and a nonobese diabetic (NOD) mouse as recipients. All five STZ BALB/c mice receiving microencapsulated islets (C57BL/6) maintained normoglycemia indefinitely. When NOD mice were used as recipients of the bioartificial pancreas, four of five grafts (islets from C3H/He) functioned for more than 80 d. Two of five NOD mice maintained normoglycemia until animals were sacrificed at 102 and 192 postoperative d. Microbeads made of commercially available agarose can effectively prolong alloislets functioning in the STZ-diabetic mouse and even in the NOD mouse (animal model of human type I diabetes) without the use of any immunosuppressive drug.

Calibration of bone mineral and heavy metal measurements using doped wall-less gel phantoms of arbitrary form

Cylindrical gel phantoms of 2% agarose concentration were used as wall-less phantoms of arbitrary shape for calibration purposes in non-destructive measurements of bone mineral and heavy metal concentration using XRF. The counts of the coherently and incoherently scattered photons with no sample present should be subtracted as “background” to get a linear relationship between the ratio of the coherently and incoherently scattered photons and the bone mineral concentration for phantoms of arbitrary diameters.

Agarose gel electrophoresis.

Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5- to 25-kb DNA fragments. The Basic Protocol in this unit can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light. Support protocols describe the use of midigels and minigels, as well as recommendations for photographing stained gels.

Detection of minisatellite sequences inPhaseolus vulgaris

An improved procedure for detecting minisatellite sequences inPhaseolus vulgaris is described. Both M13 protein III tandem repeat and the 33.15 human mini-satellite sequences revealed polymorphisms with a high number of sharp bands after digestion of genomic DNA withHae III,Hinf I, orTaq I. Improved resolution of the numerous restriction fragments detected by these probes is accomplished by one or more of the following: varying agarose concentration, using high SDS hybridization buffer, exposure of the autoradiograph without intensifying screens, and transfer of the autoradiograph to electrophoresis paper. Increased stability of the DNA-DNA hybridizations with these heterologous probes is obtained by reducing hybridization temperature. Labeling probes with the polymerase chain reaction can accentuate some restriction fragments depending upon the radiolabeled nucleotide used.

Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis.

Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase I, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid-sized DNA in mixtures with linear DNA.

Capillary electrophoresis in agarose solutions: extension of size separations to DNA of 12 kb in length.

The upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp) vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0-2.6%. It was similar in solutions at 40 degrees C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25 degrees C was inferior to that at 40 degrees C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective "equivalent radius" of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.

The substructure of agarose gels as prepared for electrophoresis

AbstractGelled agarose was observed by freeze‐fracture and electron microscopy. Aggregated double helices of agarose form a fibrous network that showed conformational variations over a range of agarose concentrations (0.3–2%). Diameters of fibers tend to increase with rising concentrations and the appearance of protuberances or cul‐de‐sacs in the network increases with falling concentration.

Time‐Resolved study of network self‐organization from a biopolymeric solution

AbstractTime‐resolved studies of network self‐organization from homogeneous solutions of the representative biostructural polymer agarose are presented. Solutions are temperature quenched and observed by several techniques. Consistent with previous suggestions by the authors, experiments at concentrations up to about 1.75% w/v provide direct kinetic evidence for the occurrence of at least two distinct processes, leading, in sequence, to self‐assembly. These are as follows: (a) a liquid–liquid phase separation of the solution occurring via spinodal demixing and resulting in two sets of regions that have, respectively, higher and lower than average concentrations of random‐coiled polymers; and (b) the subsequent 2 coils → double helix transition and accompanying cross‐linking and gelation (due to branching of double helices), occurring in the high‐concentration regions. The size of the high‐concentration regions depends upon agarose concentration and quenching temperature, and is in the range from a fraction of micrometers to a few micrometers, in agreement with earlier experiments. Bundling of the double‐helical segments is known to follow self‐assembly and can be considered as a third step (gel curing). This follows from the thermo‐dynamic instability of the helical segments in the solvent, behaving as a system of rod‐like particles connected by more or less flexible joints.The two processes leading in succession to self‐assembly are discussed in terms of a phase diagram consistent with available data. Their time scales differ remarkably. At the end of the first process, all polymers remain random coiled and freely drifting. Much later coil–helix transition is observed, always in coincidence with polymer cross‐linking and gelation. The enhancement of concentration of random‐coiled polymers in specific regions of the sol caused by spinodal demixing is thus a prerequisite for self‐assembly of these biostructural gels in the concentration interval studied. Conceptually, concentration enhancements of this type can provide a new pathway for promotion of functional biomolecular interactions even at very low average concentrations. The mechanism will work identically if the region of instability is reached by varying the polymer concentration (e.g., by biosynthesis), rather than by temperature quenching.

Microgel regions in dilute agarose solutions: the notion of non-gelling concentration, and the role of spinodal demixing

Freely drifting microgel regions are found in aqueous solutions of agarose, a representative biostructural polysaccharide, at concentrations between 0.01% and 0.05% w/v when quenched from 100°C to lower temperature. The size of these domains depends on the quench temperature and agarose concentration. The results agree with recent findings on the role that fluctuations within or close to the instability region of solution have as the initial step towards the self-assembly of supramolecular structures, and throw a new light on the notion of the lowest solute concentration needed for gelation.

Comparison between Pulsed-field and Constant-field Gel Electrophoresis for Measurement of DNA Double-strand Breaks in Irradiated Chinese Hamster Ovary Cells

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

Transient electric birefringence study of highly dilute agarose solutions

AbstractIn order to characterize the first step of agarose gelation, highly dilute solutions (2·10−3 to 0.5 g/L) have been studied by means of the transient electric birefringence technique. The field‐free decay curves of the birefringence are well described by a stretched‐exponential B(t) ≈ exp(−t/τ)β; the value of the exponent β is close to 0.5 whatever the agarose concentration. The suspended particles observed by electron microscopy present a rod‐like shape with a constant diameter (∼50 Å), without any branching; they are polydisperse with a distribution of lengths approximately exponential. The mean length of these fibers, deduced from their mean rotational diffusion coefficient, is proportional to the 0.37 power of the agarose concentration in the solution. Furthermore, these particles possess a strong permanent electrical dipole confirming the side‐to‐side arrangement of helices into bundles; this dipole is roughly proportional to the particle length, indicating a self‐similarity in the unidirectional growth of the agarose fibers, even when approaching the gelling concentration.