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At least 235 records · Page 13Linked to original sources
PubMed2000

In vitro evaluation of sol-gel processed spray dried silica gel microspheres as carrier in controlled drug delivery.

The objective of this study was to evaluate sol-gel-derived spray dried silica gel microspheres as carrier material for dexmedetomidine HCl and toremifene citrate. The drug was dissolved in sol-gel processed silica sol before spray drying with Büchi laboratory scale equipment. Microspheres with a low specific surface area were spherical by shape with a smooth surface without pores on the external surface. The particle size distribution was quite narrow. The in vitro release of toremifene citrate and dexmedetomidine HCl showed a dose-dependent burst followed by a slower release phase, that was proportional to the drug concentration in the concentration range between 3.9 and 15.4 wt.%. The release period for toremifene citrate was approximately 10 days and for dexmedetomidine HCl between 7 and 50 days depending on drug concentration. Spray drying is a promising way to produce spherical silica gel particles with a narrow particle size range for controlled delivery of toremifene citrate and dexmedetomidine HCl.

Delayed-Action Preparations↗
PubMed2004

Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels.

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

Animals↗
PubMed1995

Gene linkage of two-dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in-gel trypsin generated peptides.

The total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) where 500 micrograms was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in-gel trypsin digestion, and fractionated by reversed phase-high performance liquid chromatography (RP-HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PCD1, ENO1, ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2. The chromatograms obtained from RP-HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2-D pattern of S. cerevisiae will be obtained in sequencable amounts.

Amino Acid Sequence↗
PubMed1996

Capture in the gel: intermoleculare triplex formation during gel electrophoresis.

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence-specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel-shift assay even if their lifetime is much shorter than the time of gel electrophoresis.

Base Composition↗
PubMed1997

Detection and semiquantitation of albumin forms in fresh human plasma separated on gradient polyacrylamide gel by means of electroblotting on agarose gel matrix.

Albumin in vitro contains several molecular forms, while in vivo it exists mainly as a monomer with a small fraction of a dimer. The aim of the present work was to detect and estimate albumin forms in fresh blood samples. The available analytical methods at present are inadequate for this purpose. An improved immunoblotting method was used where plasma was subjected to electrophoretic separation on 4-25% gradient polyacrylamide gels followed by immunoblotting on agarose gel containing anti-human albumin. The interference from the huge amount of the monomer in plasma was overcome by cutting the monomer region from the polyacrylamide gel before immunoblotting. After staining of the agarose gel, it revealed the presence of seven stained bands of albumin in addition to the monomer. These bands represent albumin aggregates and complexes of varying molecular masses (112-428 kDa). These albumin forms accounted for 0.7% of the total plasma albumin and their estimated level was 30.7 mg/dL. This study shows that the native albumin in blood has several molecular forms. It is concluded that albumin in healthy human subjects may form association complexes of varying molecular masses with other macromolecules in blood and these complexes are expected to be of physiological relevance.

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis.

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris-44.5 mM boric acid-1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.

Acrylic Resins↗
PubMed2005

Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

An anomalous electrophoretic behavior of a chitinase isoform present in both grape (Vitis vinifera L.) berries and wine was observed in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. A progressive shift of the relative molecular mass M(r) of the enzyme (from approximately 30,500 up to approximately 57,700) with increasing glycol chitin concentration in the gels up to 0.1% was revealed when samples were electrophoresed under nonreducing conditions, whereas the presence of glycol chitin had no effects when samples were reduced before SDS-PAGE separation. The M(r) of other grape and wine chitinase isoforms as well as that of the chitinase from pomegranate (Punica granatum L.) fruit was unaffected by the presence of the substrate in the gel under both reducing and nonreducing conditions. Since the enzymes were inactive during the electrophoretic separation, it is likely that the retarding effect of glycol chitin observed specifically for the unreduced chitinase band from grape and wine was due to an interaction between the substrate and a chitin-binding domain different from the catalytic site, such as that typical of class I and class IV chitinases.

Chitin↗
PubMed1993

Multiple bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of proteins due to intermolecular disulfide cross-linking.

Gel electrophoresis has been used extensively as an analytical technique to check the purity and to determine the molecular weight of proteins. Improved levels of detection of proteins by silver staining of the gels have made the technique more sensitive in detecting heterogeneity. We report here some interesting observations about the anomalous behavior of some proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This report is concerned with the appearance of multiple bands, after silver staining, on the SDS-PAGE gels of proteins which are shown by HPLC to be homogeneous. We also show the reasons for the appearance of bands that originate from 2-mercaptoethanol itself.

Cross-Linking Reagents↗
PubMed1993

A gel-concentration-independent retardation detected in two fragments of the rrnB P1 promoter of E. coli using transverse polyacrylamide pore gradient gel electrophoresis.

Two fragments of the E. coli rrnB ribosomal RNA P1 promoter Upstream Activation Region exhibit a constant gel retardation, over a polyacrylamide gel concentration range of 3% to 10%. Gel retardation is usually seen to increase with polyacrylamide gel concentration as in the case of the 219 base pair Crithidia fasciculata kinetoplast DNA fragment which represents the classic case of sequence directed curvature. Computer modeling of the Upstream Activation Region fragments suggests that their unusual electrophoretic behavior can be accounted for on the basis of a "screw" model proposed by Drak and Crothers (1991, Proc. Natl. Acad. Sci. USA 88,3074-3078).

Animals↗
PubMed1992

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: an in vitro system with application to the bioartificial liver. Rat hepatocytes cultured in cylindrical collagen gels.

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.

Albumins↗
PubMed1984

Purification of monoterpenyl glycosides by gel-permeation and hydrophobic-interaction chromatography on polyacrylamide (Bio-Gel P-2).

Hydrophobic interaction of the aglycone of monoterpenyl glycosides with the polyacrylamide matrix of Bio-Gel P-2 greatly retards the elution of these substances when chromatographed in dilute aqueous sodium chloride. This hydrophobic interaction is eliminated by inclusion of 15% acetonitrile in the eluant, thereby permitting conventional gel-permeation chromatography. Combination of these techniques by sequential chromatography on the same Bio-Gel column, in the hydrophobic interaction mode followed by the gel-permeation mode, provides a simple, yet mild and highly selective procedure for the purification of monoterpenyl glycosides from crude plant extracts. Examination of the chromatographic properties of beta-D-glucopyranosides and beta-D-galactopyranosides of a number of acyclic, monocyclic, and bicyclic monoterpenols indicates that the extent of hydrophobic interaction is of diagnostic value in determining the nature of the aglycone.

Acrylic Resins↗
PubMed1984

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions.

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.

Buffers↗
PubMed1988

Use of "submarine" gel electrophoresis for running multiple two-dimensional protein gels.

An apparatus commonly used for the electrophoresis of submerged agarose gels was used to separate proteins in the second dimension, after isoelectric focusing in the first dimension. Multiple second-dimension gels were stacked one above the other and run horizontally, submerged in the sodium dodecyl sulfate-containing Laemmli buffer system. The reproducibility of the gels run under these conditions is remarkable and eliminates the need for individual vertical electrophoresis units for routine analysis. The units for submerged horizontal gel electrophoresis are easily made or are inexpensively available commercially.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Gel electrophoresis in the presence of soluble, aqueous polymers: horizontal sodium dodecyl sulfate-polyacrylamide gels.

Because little is known about the use of aqueous polymers in polyacrylamide gel electrophoresis, we undertook a feasibility study that enables the discontinuous Laemmli-formulated system of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to be performed in a horizontal format by the addition of large-sized aqueous polymers (i.e., dextrans and methylcelluloses). We studied four parameters: the cross-linking agent (bisacrylamide vs AcrylAide) and the polymer concentration, nature, and size. Three concentrations of each polymer were used. The best differentiation between the standard markers and the sharpest bands were obtained using concentrations of 2.5 and 0.06% (w/v) for Dextran T-500 and methylcellulose 4000, respectively. There was no predictable pattern to the variation in the plots of log Mr vs Rf caused by varying the concentration and length of the dextrans; however, the methylcellulose patterns suggest that gel viscosity is important. The results suggest that the combination of 0.06% methylcellulose 4000 polymers with bisacrylamide is a convenient and inexpensive means of performing flatbed Laemmli SDS-polyacrylamide gel electrophoresis.

Dextrans↗
PubMed1994

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.

Chemical Phenomena↗
PubMed1991

Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection.

Denaturing gradient gel electrophoresis (DGGE) is increasingly being utilized in mutational detection, both in characterization of variations in genomic DNA and in the generation of mutational spectra after in vitro and in vivo mutagenesis. The basis for this electrophoretic separation technique is strand dissociation of DNA fragments in discrete, sequence-dependent melting domains followed by an abrupt decrease in mobility. We have modified the DGGE by using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments. This leads to increased resolution of mutants as fragments differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains. We have used this technique to separate 6 out of 7 exon-3 hypoxanthine phosphoribosyltransferase (HPRT) mutants while using conventional DGGE we were only able to separate 3.

Animals↗
PubMed1989

Two-dimensional gel electrophoresis of RNase T1 resistant oligonucleotides of flavivirus RNA using ultrathin gels.

A convenient method employing a commercially available apparatus for two-dimensional gel electrophoresis of RNase T1 resistant oligonucleotides using ultrathin gels has been developed. The methodology overcomes problems commonly associated with the establishment of good, bubble-free, fusion of the first and second dimension gels. The use of ultrathin gels results in autoradiograms with well resolved oligonucleotide spots.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics.

Comparing different proteomes by classical two-dimensional electrophoresis is challenging and often complicated by substantial gel-to-gel variation. Separating two or more protein samples labelled with different fluorescent dyes in one single gel, as in two-dimensional difference gel electrophoresis, reduces this variability considerably. Recent technological innovations, specifically the introduction of a pooled internal standard, even further improve the quantification accuracy and statistical confidence of this method. In addition, decreasing the sample complexity by one of several protein or organelle fractionation procedures increases the number of spots investigated by this protein differential display methodology.

Animals↗