Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

ABSTRACT: Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

SUBMITTER: Rice KG

PROVIDER: S-EPMC1148026 | biostudies-other | 1987 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis.

Project description:Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.

| S-EPMC2860026 | biostudies-literature

Separation of radiolabelled glycosaminoglycan oligosaccharides by polyacrylamide-gel electrophoresis.

Project description:Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.

| S-EPMC1144099 | biostudies-other

Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.

Project description:Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.

| S-EPMC1144100 | biostudies-other

The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis.

Project description:1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.

| S-EPMC1270235 | biostudies-other

A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.

Project description:BackgroundSulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes.ResultsA universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. This assay has been successfully applied to substrates as small as alpha-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of +/- 0.2 of the actual relative mobilities.ConclusionThe versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

| S-EPMC2834601 | biostudies-literature

Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.

Project description:In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

| S-EPMC3755479 | biostudies-literature

Resolving mitochondrial protein complexes using nongradient blue native polyacrylamide gel electrophoresis.

Project description:Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomics analysis of high-molecular-weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this article, we present an alternative BN-PAGE method that uses highly porous, nongradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I to V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be detected simultaneously on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, nongradient gel electrophoretic approach that should be useful in functional proteomics studies.

| S-EPMC2795571 | biostudies-literature

Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane.

Project description:A new method that we have called 'oligosaccharide mapping' is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources.

| S-EPMC1149043 | biostudies-other

GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis.

Project description:Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.

| S-EPMC383056 | biostudies-literature

Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices.

Project description:New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this "open" fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photopattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.

| S-EPMC6777840 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data