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Results for “Chromatography, Affinity”

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At least 289 records · Page 16Linked to original sources

PubMed1991

High-performance affinity chromatography of NADP+ dehydrogenases from cell-free extracts using a nucleotide analogue as general ligand.

An epoxy-activated silica column (50 cm x 0.45 cm I.D.) was derivatized with 8-[6-aminohexyl)amino]-2'-phosphoadenosine-5'-diphosphoribose; the bound ligand concentration was 11.4 mumol/g of dry silica, and the useful loading capacity was 2.3 mg of glutathione reductase. The new high-performance liquid chromatographic column specifically retained NADP(+)-dependent enzymes, which were quantitatively eluted specifically by NADP+ or, with better resolution, by potassium chloride. The new high-performance liquid chromatographic support was applied to the purification of glutathione reductase and glucose-6-phosphate dehydrogenase from cell-free extracts of baker's yeast, fish liver and rabbit hemolysates, with high recoveries and excellent purification factors.

Animals↗

PubMed1992

Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins.

The chromatographic behaviour of phosphoamino acids, phosphopeptides and phosphoproteins and their non-phosphorylated counterparts was studied on Fe(III)-Chelating Sepharose and Fe(III)-Chelating Superose. The phosphorylated compounds, in contrast to their non-phosphorylated or dephosphorylated counterparts, adsorb to immobilized iron(III) ions at pH 5.5 and can be desorbed by an increase in pH. Phosphoamino acids were eluted at pH 6.5-6.7, whereas monophosphopeptides and phosphoprotamine eluted in the pH range 6.9-7.5. Molecules possessing clusters(s) of carboxylic groups are weakly retained (gamma-carboxyglutamic acid, Ala-Ser-Glu5) or bound (polyglutamic acid, beta-casein) to the immobilized iron(III) ions at pH 5.5. Dephosphorylated beta-casein was desorbed at pH 7.0, whereas for elution of native (non-dephosphorylated) beta-casein, phosphate buffer of pH 7.7 was required. The homopolymer of polyglutamic acid was desorbed in the pH range 6.0-6.3, whereas copolymers of glutamic acid and tyrosine require pH 7.0-7.3 or even phosphate buffer at pH 7.7 for elution.

Amino Acid Sequence↗

PubMed1992

High-performance affinity chromatography of DNA. II. Porosity effects.

A DNA-silica, (dT)18-silica, was prepared and used in a study of the chromatography of the oligonucleotide, (dA)18, based upon base pairing. It was shown that hybridization efficiency did not depend upon flow-rates up to 2 ml/min for the small columns (22 x 2 mm) used. As increasing amounts of (dA)18 were loaded onto the columns, the columns were found to saturate at a well defined capacity that was always less than the amount that theoretically could have been bound. Maximum capacity was achieved whenever the loading temperature was at least 20-25 degrees C below the temperature at which the loaded oligonucleotide would elute. The effects of porosity on both coupling efficiency and capacity were measured and suggest that pore sizes in the 300-500 A range are most appropriate for this form of chromatography.

Chromatography, Affinity↗

PubMed1977

Affinity chromatography of antiserum to a gram negative organism.

An immunoadsorbent method is described for the purification of antibody directed against gram negative bacteria. Two anion exchange materials were compared for their ability to immobilize whole cells of Veillonella alcalescens, a gram negative oral bacterium. Purified antibody preparations were applied to the columns and subsequently eluted with various combinations of desorbing buffers. The quantity of recovered antibody was measured and its activity assayed, using a microagglutination technique. The highest levels of protein and specific antibody activity were recovered from Dowex-1-acetate columns desorbed by a combination of borate buffer pH 10 and 3 M KSCN pH 6, followed by levels of specific antibody activity obtained from a DEAE cellulose column desorbed by glycine--HC1, PBH 2.3.

Chromatography, Affinity↗

PubMed1980

Isolation of subclasses of human IgG with affinity chromatography.

A method is described for the isolation of subclasses of human IgG. Two myeloma proteins, after isolation by DEAE-Sephadex chromatography, were further purified on subclass-specific anti-IgG1 and 2 Sepharose 4B columns. Contaminating IgG1 and 2 were removed from the isolated myeloma proteins.

Animals↗

PubMed1993

Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles.

IgG from patients with rheumatoid arthritis (RA) is abnormally glycosylated in the Fc region, with sialic acid and galactose levels lower than normal. Protein G and DEAE purify populations which are differentially glycosylated. Significantly increased exposure of sialic acid was detected in normal IgG compared with that of RA IgG when ion exchange was used to prepare samples. However, when the same samples were prepared using protein G, no difference in the detection of sialic acid was seen between the two groups. When examining the heavy chain of IgG, more sialic acid, galactose and N-acetylglucosamine were detected in DEAE purified IgG compared with that prepared by protein G Detection of sialic acid and N-acetylglucosamine was also increased on light chains from IgG prepared by ion exchange chromatography. Since this occurs notably on rheumatoid light chains it would appear that this arrangement would contribute to the overall glycosylation changes in IgG. In the case of molecules lacking galactose the discrimination between the RA and normal IgG is significantly improved when ion exchange chromatography is used. Since differentiation between disease and normal groups relies on the purification technique used, we recommend that more than one method is employed before undertaking an analysis of glycosylation changes.

Arthritis, Rheumatoid↗

PubMed1979