Digitonin is a mild detergent that permeabilizes plasma membranes selectively due to their high cholesterol content, whereas mt-membranes with lower cholesterol content are affected only at higher concentrations. Digitonin is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see: SUIT-010 O2 ce-pce D008). Abbreviation: Dig Reference: Pesta 2012 Methods Mol Biol, MiPNet09.12 O2k-Titrations ...
Digitonin permeabilized cell import assay. Complete import assays were carried out using HeLa cells permeabilized by incubation with digitonin. Cells were incub
Calcitonin gene-related peptide (CGRP) receptors were solubilized from rat cerebellum membranes in an active, stable, and guanine nucleotide-sensitive form by using digitonin. Nearly 90% of membrane CGRP receptors and 50% of membrane protein were solubilized by digitonin treatment of cerebellum membranes. Binding of 125ICGRP to soluble receptors was specific, saturable, of high affinity, and reversible. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites with a Kd of 178 +/- 42 pM and a Bmax of 201 +/- 17 fmol/mg of protein. Binding of 125ICGRP to soluble receptors was inhibited nearly 60% by guanosine-5-O-(3-thio)triphosphate (GTP gamma S) (100 microM), suggesting coupling of receptors with guanine nucleotide-binding proteins (G proteins) to form high affinity binding sites. Antiserum against the amino-terminal region of Gs alpha immunoprecipitated a significant portion of soluble CGRP receptors, indicating association of receptors with Gs ...
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Although we have shown that bivalent TCR/CD3 are present among digitonin-solubilized complexes, we do not know the proportion of complexes that they represent. One focus of future experiments must be to develop other methods that would permit a quantitative estimate of the prevalence of bivalency among all TCR/CD3 complexes. However, despite this current technical limitation, the data in Fig. 7 imply that the proportion of bivalent complexes is sufficiently high to impact the outcome of the pMHC-Ig fusion protein binding assay performed. Thus, based on the assumption that this assay reflects a true potential for functional impact, we speculate that it is likely that a biologically significant number of TCR/CD3 complexes display bivalency.. It is not known which motifs might specifically interact to compose a bivalent complex. Even in the standard monovalent model of TCR/CD3, the interactions between subunit dimers (αβ/εγ/εδ/ζζ) that compose the multiprotein complex are not fully ...
No, what Im saying is that NP40 and Triton X-100 are both too strong and they might break down any cell component. You need to study all these detergents very well. There are plenty of textbooks describing each one of them. For fractionation usually people use milder detergents. I found digitonin a very reliable detergent. Last time I even tried Tween 20 but I couldnt fractionate well. However, it depends on the protocol too. I would guarantee Afshin Samalis method. It really works, and it fractionates fast.. ...
Custom Filling & Packaging. Custom Filling & PackagingWe understand how important it is to fill each and every container to your exact specification to avoid waste and ensure consistency for ... [more]. Company. Products & MarketsBiosynth innovates, develops and produces precious reagents and biochemicals for the life science and pharmaceutical industries.Our main activities involve the manufacture of substances ... [more]. Innovation in Microbiology. Innovation in MicrobiologyWhile we keep our proprietary processes as trade secrets, we file patents for our unique application developments. Examples are:A. Aldol® Novel Indicator Platform: ... [more]. Biosynth News. NewsAugust 2017The headliner of this issue of the Biosynth eNews is Digitonin. We also introduce FLsharp-Phosphate, a fluorescent substrate that creates super sharp localized fluorescence ... [more]. Chemical Roles. Chemical ClassificationsChemical Roles: This section classifies entries on the basis of their roles played within a ...
The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting a regional pattern in the intact liver and a varied response to phenobarbital in isolated cultured hepatocytes. We report that P450 2B1/2 immunostaining of hepatocytes isolated from the perivenous liver region and cultured in the presence of phenobarbital is much stronger than that of cells identically treated but isolated from the periportal region. P450 2B1 mRNA, quantified by a sensitive and specific RNAase protection assay, is also preferentially induced in perivenous hepatocytes, demonstrating that the difference in induced expression is at the pretranslational level. Our results suggest that perivenous and periportal hepatocytes are differentially imprinted to retain regiospecific factors governing their inducibility after isolation.. ...
Nonnenmacher, Y., Palorini, R., dHerouël, A. F., Krämer, L., Neumann-Schaal, M., Chiaradonna, F., Skupin, A., Wegner, A. & Hiller, K. Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization. Metabolic Engineering 43, 147-155 (2017).. Mak, T. W., Grusdat, M., Duncan, G. S., Dostert, C., Nonnenmacher, Y., Cox, M., Binsfeld,C., Hao, Z., Brüstle, A., Itsumi, M., Jäger, C., Chen, Y., Pinkenburg, O., Camara, B.,Ollert, M., Bindslev-Jensen, C., Vasiliou, V., Gorrini, C., Lang, P. A., Lohoff, M., Harris,I. S., Hiller, K. & Brenner, D. Glutathione Primes T Cell Metabolism for Inflammation. English. Immunity46, 675-689 (2017).. Meiser, J., Krämer, L., Sapcariu, S. C., Battello, N., Ghelfi, J., DHerouel, A. F., Skupin,A. & Hiller, K. Pro-inflammatory Macrophages Sustain Pyruvate Oxidation throughPyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression. en. J. Biol. Chem. 291, 3932-3946 (2016).. Michelucci, A., ...
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In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional ...
The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated ...
A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed dual-digitonin-pulse perfusion. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific ...
149. Okuda K., Li L., Kudlicka K., Kuga S., and R. M. Brown ,Jr. 1993. ß-glucan synthesis in the cotton fiber. I. Identification of ß-1,4- and ß-1,3- glucans synthesized in vitro . Plant Physiol. 101: 1131-1142. 149. Abstract In vitro -glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Gossypium hirsutum) fiber cells. The reaction mixture favoring ß-l,4-glucan synthesis included the following effectors: Mg2+, Ca2+, cellobiose, cyclic-3:5-GMP, and digitonin. The ethanol insoluble fraction from this reaction contained ß-1,4- glucan and ß-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the ß-1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated product favoring ß-1,4-glucan synthesis strongly resembled that of cellulose II. On the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan product was found to be exclusively ...
The marker enzymes, alanine aminotransferase and glutamine synthetase, verified that enriched periportal and perivenous hepatocytes were isolated from different lobular origin within the rat liver. Our finding that alanine aminotransferase activity was higher in periportal hepatocytes (with a ratio of 1.9 for periportal to perivenous activities, Table 1) was in agreement with histochemical evidence on the enzyme in rat liver (Gorgens et al., 1988) and what was observed in other studies (Sillau et al., 1996; Tosh et al., 1996). The dramatic difference in glutamine synthetase activity between the perivenous and periportal regions (Table 1) was also shown by Stoll et al. (1991) and provided evidence on the successful preparation of enriched populations of periportal and perivenous cells. In addition, glutamate uptake, mediated by the sodium-dependent transporter, System G, in the perivenous region (Stoll et al., 1991) as well as the sodium-independent system in both periportal and perivenous ...
Cultures of bovine adrenomedullary chromaffin cells accumulated 1-methyl-4-phenylpyridinium (MPP+) in a time- and concentration-dependent manner by a process that was prevented by desmethylimipramine. The subcellular localization of the incorporated [methyl-3H]MPP+ was examined by differential centrifugation and sucrose density gradient fractionation and was found to be predominantly colocalized with catecholamines in chromaffin vesicles, and negligible amounts were detected within the mitochondrial fraction. When chromaffin cell membranes were made permeable with the detergent digitonin in the absence of calcium, there was no increase in the release of [3H]MPP+, indicating that there is negligible accumulation of the neurotoxin in the cytosol. Simultaneous exposure to digitonin and calcium induced cosecretion of MPP+ and catecholamines. Stimulation of the cells with nicotine released both catecholamines and MPP+ at identical rates and percentages of cellular content in a calcium-dependent ...
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Pellicular fragments were isolated from ethanol-fixed cells of the holotrichous ciliate Tetrahymena pyriformis by the action of digitonin. The isolated pellicles were further fragmented and the basal bodies of the cilia isolated from them by three methods. The preparations, examined in the electron microscope as embedded sections or negatively stained samples, consisted mainly of somewhat deformed pellicular material, the bulk of which was basal body. DNA was determined by the diphenylamine method and by reaction with DNase, and RNA, by the orcinol method. Nucleic acids were isolated by phenol extraction and analyzed spectrophotometrically and by reaction with RNase. The assays indicated 1.2 to 2.6 per cent RNA, similar to previously published work, but only 0.0 to 1.0 per cent DNA, near enough the sensitivity limits to render the presence of DNA in the preparations uncertain. Although the isolation procedure removed nuclear contents and ribosomes, the nucleic acids could still be a residual ...
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Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH ...
We have recently described the affinity chromatography purification of the turkey erythrocyte beta-adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10-fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine-sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l-epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4-6 pmoles of 3H-dihydroalprenolol binding sites per mg of membrane protein. A single step of affinity chromatography on alprenolol-sepharose of the soluble digitonin extract resulted in an additional 1,000-fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane ...
The presence on carbon-3 of a free hyclroxyl group that may be esterified is a common characteristic of the known sterol inhibitors of cholesterol absorption. The 30-carbon sterols found in wool fat possess such a group, but differ from the previously demonstrated inhibitors of cholesterol absorption in that they do not form insoluble precipitates with digitonin. To determine whether these substances would influence cholesterol absorption, "isocholesterol," a mixture of 30-carbon wool fat sterols, has been fed to the albino rat alone and in combination with cholesterol.. ...
But, of course, glucosides are glycosides.. Glycosides are constituents bound to a sugar, and in a glucoside the sugar is glucose. In a fructoside the sugar is fructose, in a galactoside the sugar is galactose (dunno how youd get milk sugar into a plant, though), and so on.. Because of that sugar glycosides usually start working in the gut, after the sugar has been stripped away.. Glycosides are water- and alcohol-soluble.. The cyanoglycosides are found in most if not all rose family plants. You get cyanide from cyanoglycosides; theyll stop you breathing if you ingest large enough amounts of them.. Youll find heart glycosides in foxglove (Digitalis spp.) and a lot of other plants which act on the heart. The glycosides digitoxin, digitalein, digitonin, digitalin and so on are cumulative and toxic; they work in doses rather too close to the deadly dose, and they will kill people who regularly take them -- if these folks suddenly get much weaker. The thing is, though, that without these toxic ...
The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase. ...
Existing biozonation schemes for the Niger Delta are mainly qualitative with zonal intervals too large to record subtle events. This has made it necessary to look for additional palynological events to enable recognition of shorter, more refined interval zones to improve stratigraphical definition. Hitherto unrecognised occurrence trends of palynomorphs were discovered and used to construct a new zonation scheme that can be applied in the offshore delta area. Late Miocene to Early Pleistocene sediments have been divided into five principal assemblage zones: FF1, Anthoceros abundance zone; FF2, Elaeis guineensis-Echiperiporites icacinoides zone; FF3, Lycopodium-Retibrevitricolporites obodoensis/protrudens zone; FF4, Cyperaceae abundance zone; and FF5, Echitriletes pliocenicus-Podocarpus milanjianus zone. The zones are further subdivided into 16 sub-zones based on quantitative events with some having finer subdivisions into (a) and (b). Examples of the zonations applied to three exploration wells ...