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PubMed2000

Gel-filtrated fractions of alveolar bone extract contain factors promoting cell attachment and a mitogenic effect on periodontal ligament fibroblasts.

The purpose of this study was to investigate the effect of acetic acid-extracted bone proteins on human periodontal ligament fibroblasts (hPF) with respect to mitogenic and cell attachment promoting activity. Alveolar bone was harvested from healthy donors and subjected to 0.5 M acetic acid extraction, dialysis and lyophilization, and gel filtration. Promotion of cell attachment and stimulation of DNA synthesis by the crude extract and gel-filtrated fractions were studied in cultured hPE Many protein components, varying in molecular weight from 10-14 to 120 kDa, were detectable in 10% SDS-PAGE of the extract. Gel filtration of bone extract disclosed four fractions with molecular weights of 55, 34, 29 and 19-20 kDa. Both the 34 and 55 kDa fractions at a concentration of 5 microg/ml, but not the 29- or 19-20 kDa fractions, were found to promote cell attachment while only the 55 kDa fraction (5 microg/ml) stimulated DNA synthesis of hPF, Both mitogenic activity and the promotion of the cell attachment by gel-filtrated active fractions were resistant to thermal treatment (70 degrees C) and pH (4 to approximately 8) changes. These findings suggest that acetic acid extract of alveolar bone may contain components which are capable of modulating cell attachment and mitogenesis of hPF.

Acetic Acid↗
PubMed1984

Comparison of gel filtration and ammonium sulphate precipitation in the purification of diphtheria toxin and toxoid.

Crude diphtheria toxin and toxoid were subjected to purification by gel filtration and stepwise ammonium sulphate precipitation. The various fractions obtained by the purification procedures were studied by immunological methods. A high molecular weight fraction of glycoprotein nature was present in all of the crude preparations studied. The fraction was antigenically non-identical with the real toxin or toxoid and did not have its origin in the culture medium. It showed a long flocculation time when tested against equine diphtheria toxoid antiserum. The fraction could be removed from the crude preparations by gel filtration or by precipitation with 21% (w/v) ammonium sulphate. When comparing toxoids purified by each of these methods, the method of gel filtration resulted in a somewhat higher degree of purity, suggesting that this method would be more suitable than the AS precipitation method for the purification of diphtheria toxoid.

Ammonium Sulfate↗
PubMed1979

A gel filtration approach to the study of ribosome-membrane interactions.

1. Gel filtration on agarose can be used to investigate ribosome-membrane interactions without exposing the materials to the high, and possibly perturbing, hydrostatic pressures experienced during centrifugation procedures to separate free ribosomes from membrane vesicles. 2. After treatment of microsomes with degranulating agents, degranulated membranes are isolated from Sepharose 2B columns at the void volume, while displaced ribosomes elute at the total column volume. This provides a convenient method for monitoring degranulation in vitro. 3. Centrifugation of rough microsomes or ribosomes into dense pellets or layers, followed by resuspension, leads to preparations which will not pass rapidly or quantitatively through Sepharose 2B columns. 4. Methods are described for the isolation of degranulated microsomes and ribosomes which are eluted rapidly from Sepharose 2B at the void volume and total column volume, respectively. These materials are suitable for the investigation of ribosome-membrane binding in vitro, using a gel filtration separation to monitor binding. 5. Incubation of 3H-labelled ribosomes with degranulated microsomes in vitro, leads to specific binding, demonstrated by the elution of the bound ribosomes at the void volume.

Animals↗
PubMedDate not supplied

[Study of the specific anti-influenzal activity of human respiratory tract secretions and their fractions isolated by means of gel filtration].

It was found that in gel filtration of a mixture of the human nasal cavity and salivary gland secretions on Sephadex G-200, three protein peaks were regularly releaved. According to the results of disk electrophoresis, the original secretion mixture consists of 11--13 fractions. The material of peak No. 1 is found in one di-fuse band on the cathode end of gel. Peak No. 2 contains 5--7 fractions and eluate No. 3 contains 3 fractions. The gel precipitation test with monospecific rabbit antisera to human IgM, IgG and IgA demonstrated IgA to be present in the first peak.

Antibody Specificity↗
PubMed1979

Gel filtration in Sephadex G200 for the control of preparations of human immunoglobulin.

Gel filtration in Sephadex G200 has been used in the quality control of normal and specific immunoglobulins at the Blood Products Laboratory, Elstree, for more than eleven years. Eluting from a 140 cm column, polymerised and aggregated protein is excluded by the gel, followed in order of elution by fractions containing the IgG dimer and the monomer. After these, smaller protein impurities such as albumin are eluted and then fragments from the proteolytic breakdown of IgG. On a single chromatogram, possibly five distinct peaks may be recorded and their components quantified. Gel filtration in Sephadex G200 provides an alternative to sedimentary boundary ultracentrifugation, by which fragmentation of IgG can be more readily observed. In addition, the fraction containing the polydisperse aggregates excluded by the gel provides an index of adverse treatment of immunoglobulin during preparation and storage.

Chromatography, Gel↗
PubMed1976

Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities.

Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed.

Agglutinins↗
PubMed1987

Varicella-zoster virus IgM antibody fraction separated by minicolumn gel filtration in serodiagnosis of acute infection.

Gel filtration through Sephadex G-200 or Ultrogel AcA 34 minicolumns was applied to isolate IgM antibodies from sera of patients with varicella and herpes zoster (HZ). Serum samples were stained with Sudan III and Evan's Blue in order to monitor the separation process visually when running several columns at the same time. The presence of specific IgM antibodies was demonstrated by enzyme linked immunosorbent assay (ELISA). Rapid separation of stained sera by diffusion chromatography on minicolumns with successive determination of specific IgM antibodies in ELISA proved suitable for serological diagnosis of varicella, whereas the significance of IgM-specific antibody response in HZ seems uncertain.

Acute Disease↗
PubMed1990

Effect of experimental conditions on the appearance of distinct forms of placental glucosylceramidase: use of gel filtration analysis as a means of ascertaining their occurrence.

We have found that, under some experimental conditions, the placental glucosylceramidase shows an anomalous behaviour on gel filtration chromatography. At pH 5.6, the optimal pH of the enzymatic assay, the purified enzyme remains bound to either Superose 6 or TSK-40-XL HPLC columns, while the interaction of the crude glucosylceramidase contained in the water extract of the lysosome-mitochondrial fraction of placenta with the two HPLC gel matrices is much weaker. The quite different behaviour of the crude compared to the purified enzyme may be explained by the formation in the crude preparation of associated form(s) of glucosylceramidase with suitable endogenous compound(s), which compete with the gel matrices for the binding to the enzyme. The most likely one component of the enzyme complex is the placental activating factor, previously reported by us (Vaccaro et al. (1985) Biochim. Biophys. Acta 836, 157-166), as indicated by the negligible stimulation of the crude enzyme activity on addition of the factor, either before or after passage through the HPLC columns. On the assumption that the behaviour of crude glucosylceramidase on gel filtration becomes similar to that of the purified enzyme when its interaction with endogenous substance(s) is impaired, we have identified some conditions which prevent the formation of the enzyme associated form(s): (a) the addition of guanidine chloride (0.2 M), a cahotropic agent, to the crude preparation; and (b) the increase of pH up to 8. In conclusion, taking advantage of the anomalous behaviour of glucosylceramidase on gel filtration chromatography, evidence has been obtained that placental glucosylceramidase may occur under several forms which had not been previously reported; a difference in experimental conditions can promote the formation of one or another form, by possibly affecting the composition and/or the stoichiometry and/or the stability of the enzyme complex.

Chromatography, Gel↗
PubMed1993

Analytical gel filtration of dextran for the study of the glomerular barrier function.

Analytical gel filtration was used for the study of molecular size distribution of clinical dextran in serum and urine for the purpose of evaluations of changes in the human glomerular barrier function. The column was calibrated in terms of solute size using a simple and accurate technique recently described. Only one sample of a dextran possessing a broad molecular mass distribution was necessary for the calibration procedure and the calculations were performed using an ordinary spreadsheet. The accuracy of the calibration, as evaluated by protein samples, is better than 95%. The simplicity makes the method suitable for use in laboratories not normally specializing in analytical gel filtration. Calibration in terms of size is preferably done with respect to viscosity radius to obtain relevant information about the permeability of dextran into porous membranes.

Chromatography, Gel↗
PubMed1979

Use of electrophoresis and gel filtration to characterize human pituitary somatotropin preparations after storage.

Fresh and stored preparations of human pituitary somatotropin examined by different polyacrylamide gel electrophoresis techniques and by gel exclusion chromatography (Ultrogel) clearly demonstrated that the hormone undergoes a structural alteration during storage for 3 years. The formed aggregates were readily quantitated from the gel filtration profile and from polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). According to gel filtration the amount of aggregates in fresh and stored hormone was determined to be 2 and 10%, respectively, while SDS-PAGE indicated 6 and 14%.

Chromatography, Gel↗
PubMed1988

Association-dissociation studies of bovine and rat liver glutamic dehydrogenase by high-performance liquid chromatography gel filtration.

The concentration-dependent association-dissociation tendency of purified bovine liver and rat liver glutamic dehydrogenase (GDH) has been demonstrated by high-performance liquid chromatographic gel filtration. In the concentration range of 100 to 1.0 micrograms bovine GDH/ml molecular species ranged from dimer and unimer to subunimeric forms. The dissociation process of the unimeric hexapeptide, consisting of six polypeptide chains, to the subunimeric tripeptide, consisting of three polypeptide chains, was irreversible without added ionic support, but reversible with added ionic support. In dilute Tris-HCl bovine liver GDH was dispersed to subunimeric sizes. Increasing the ionic strength in 20 mM phosphate as the mobile phase increased dissociation to a subunimeric tripeptide while sustaining as much as 80% of its activity. Activity of a eluting subunimer was verified by the inclusion of reaction substrates (NAD and glutamute) in the mobile phase and quantification of reaction products (NADH) in chromatograms. Gel filtration of GDH in the presence of GTP with NADH rendered a subunimeric tripeptide, largely independent of ionic strength or GDH concentration. Rat liver GDH, differing from bovine liver GDH, was dissociated by gel filtration to an active tripeptide independent of ionic or buffer conditions.

Adenosine Diphosphate↗
PubMed2002

[The renaturation and purification of RGD-staphylokinase by gel filtration].

A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E. coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation, After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demonstrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.

Chromatography, Gel↗
PubMed1981

Separation of low molecular weight RNA species by high-speed gel filtration.

By employing a high-speed gel filtration method using a column of silica-based aqueous gel, TSK-GEL G 300 SW, the three low molecular weight RNA species, 5.8S and 5S rRNA, and tRNA, were clearly separated from each other and from high molecular weight RNA species in 200 mM sodium phosphate buffer, pH 7.0, containing 0.1% SDS. Excellent reproducibility and resolution were obtained under these conditions, about 30 min being required for a single run. Furthermore, the 5.8S rRNA from rat liver cells was eluted at a different position compared with that of the yeast, Saccharomyces cerevisiae, although they have the same nucleotide chain length but at different sequence. The 5.8S rRNA from the cellular slime mold, Dictyostelium discoideum, was eluted at the same position as that of yeast.

Animals↗
PubMed1981

Gel filtration of urine: a method to detect nephrotoxicity in rats.

Sephadex G-100 gel filtration of urine from male Wistar rats revealed 3 protein peaks: peak I (eluted with the void volume of the column), peak II (Mr between 67 000 and 43 000), and peak III (Mr about 13 700). Nephrotoxic compounds were given as a single i.p. injection. Peak I, and especially peak II were significantly increased in 24-h urine samples from rats receiving mercuric acetate (1.58 mg/kg), mercuric trifluoroacetate (MTFA) (2.22 mg/kg), sodium ethylmercurithiosalicylate (EMTSA) (20.2 mg/kg), sodium tetrathionate (250 mg/kg), ammonium fluoride (18.5 mg/kg), paramomycin sulfate (800 mg/kg), ochratoxin A (5 mg/kg) and cis-platinum (4 mg/kg). It is concluded that gel filtration of urine can be used as a method to detect nephrotoxicity in rats.

Animals↗