SEARCH · PubMed Health

Results for “Chromatography, Affinity”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed1987

Dual-column determination of albumin and immunoglobulin G in serum by high-performance affinity chromatography.

High-performance affinity chromatography was used for the simultaneous determination of albumin and immunoglobulin G in serum. Two columns in series, the first containing immobilized anti-albumin antibodies and the other containing protein A for binding immunoglobulin G, were eluted separately at pH 3 by means of a column-switching system. This method gave results in good agreement with commercially available methods, while requiring only 2 microliter of serum and 6.0 min per cycle. It was shown that albumin and immunoglobulin G were selectively retained, with little interference from other components, including immunoglobulins A and M.

Chromatography, Affinity↗

PubMed2001

Perspectives of immobilized-metal affinity chromatography.

Immobilized Metal-Affinity Chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface-exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are discussed in this article. Advantages of the method and new prospects are described as well as the problems and concerns that appear when the method is to be used for production of pharmaceutical-grade proteins.

Binding Sites↗

PubMed2001

Polymer versus monomer as displacer in immobilized metal affinity chromatography.

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.

Chromatography, Affinity↗

PubMedDate not supplied

Screening of protein-ligand interactions by affinity chromatography.

This paper examines affinity chromatography (AC) as an alternative tool for the determination of protein-ligand interactions for the particular case in which the ligand is the same protein. The methodology is less labor-intensive and more sample-efficient than traditional methods used to measure the second virial coefficient (B(22)), a parameter commonly used to evaluate protein-protein interactions. The chromatographic capacity factor (k') was studied for lysozyme and equine serum albumin for a wide range of experimental solution conditions such as crystallizing agent concentration, protein concentration and pH. Parallel experiments using AC to determine k' and static light scattering (SLS) to determine B(22) showed that the two parameters were highly correlated. Two different column volumes ( approximately 1 and approximately 0.1 mL) were tested and gave essentially the same values for k', showing the feasibility of miniaturization.

Chromatography, Affinity↗

PubMedDate not supplied

[Affinity chromatography of nucleoprotein complexes].

Affinity sorbent containing cellular membranes of the cerebral cortex was obtained by means of an inclusion into porous matrix of polyacrylonitrile. Sorption of model and natural nucleoprotein complexes on the obtained sorbent was studied by the method of affinity chromatography. Distribution coefficients were determined. Natural nucleoprotein complexes isolated from brain possess a greater selectivity of binding to the cellular membranes of the cerebral cortex than that from liver.

Acrylic Resins↗

PubMedDate not supplied

[Purification of Bacillus mesentericus proteolytic enzymes by affinity chromatography].

By means of affinity chromatography on Ovomucoid-Sepharose two proteinases hydrolyzing casein and elastin were isolated from the supernatant of the Bacillus mesentericus culture medium. The activity yield of proteinases was 100%. The characteristics of the purified enzymes were studied. It is demonstrated that B. mesentericus possesses several proteinases.

Bacillus↗

PubMed1985

UV-absorbance profile of human leukocytic ultrafiltrate after affinity chromatography on immobilized m-aminophenyl boronic acid: implication for transfer factor purification.

The UV-absorbance of the lysed human leukocyte ultrafiltrate (LLU) was investigated by Sephadex G-15 gel permeation chromatography before and after affinity chromatography on immobilized m-aminophenyl boronic acid. This biospecific fractionation caused substantial changes in 260/280-nm absorbance ratio of individual peaks as revealed by Sephadex G-15 gel permeation chromatography. While the material which had passed through the affinity support unretarded (W) appeared to be partly or completely deprived of peak IV, peaks IV and V were fairly enriched in the retarded material (R). This material, on the other hand, nearly completely missed peaks III, VI and VII. Peak III was copiously represented in material W. Boronate affinity chromatography caused enrichment in the protein content as well as in the content of purine/pyrimidine-bound ribose of material R, i.e. cis-diol group containing (RNA-like) material, considered to represent functional transfer factor activity. The described findings further support the use of immobilized derivatives of boronic acid in the separation of coplanar cis-diol group containing compounds from other constituents of LLU.

Boronic Acids↗

PubMed1979

Isolation and immunochemical characterization of antibodies from the sera of cancer patients which are reactive against human melanoma cell membranes by affinity chromatography.

Immunoglobulins were isolated by affinity chromatography from sera of two patients with melanoma, one with sarcoma, and one with carcinoma. The affinity columns were prepared by covalently linking the membrane-rich fraction of biopsied melanoma cells to cyanogen bromide-activated agarose beads. The membrane-rich fractions were prepared by two methods: (a) hypotonic cell lysis, and (b) homogenization and differential centrifugation. Melanoma sera were autologous to melanoma membrane preparations. The isolated immunoglobulins showed immunoreactivity against antigens prepared from melanoma, sarcoma, and carcinoma cells by complement fixation but not against antigens prepared from normal human liver and lung tissues. Absorption of the isolated immunoglobulins with rabbit anti-human immunoglobulin immunobeads resulted in complete elimination of the complement-fixing antibody titer in one instance, whereas reduction occurred in other samples. Similar absorption with rabbit anti-human immunoglobulin M immunobeads resulted in reduction, but not complete elimination, of the antibody titers against target tumor cell preparations. These results suggest the presence of immunoreactive immunoglobulin G in all immunoglobulins and immunoglobulin M in some. Absorption of the isolated immunoglobulins with cultured sarcoma cells reduced but did not completely abolish antibody activity against autologous or allogeneic melanoma target antigen, whereas it did completely abolish activity against sarcoma target antigen. However, absorption with cultured allogeneic melanoma cells abolished the antibody activity against melanoma as well as sarcoma target antigens. The antibody titers of the isolated immunoglobulins were not affected by absorption with cultured lymphoblastoid cells. Since cultured melanoma and sarcoma cells were known to contain oncofetal antigen(s), these results suggest that the isolated immunoglobulins from cancer sera by melanoma membrane affinity chromatography were of at least two specificities: (a) antioncofetal; and (b) antitumor associated. The former group may be comprised of antibody to cross-reactive antigens associated with different histological types of tumors. However, it was apparent that a portion of the antibody activity was against common tumor-associated antigen(s). These results provide further evidence for the presence of common antigen(s) associated with biopsy specimens of human malignant melanoma.

Adult↗

PubMed1987

Affinity chromatography of glycopeptide antibiotics.

An affinity support was designed to facilitate the isolation and purification of glycopeptide antibiotics by mimicking their known affinity for the bacterial cell wall. Members of this class of antibiotics inhibit peptidoglycan biosynthesis by specifically binding to pentapeptide precursors terminating with L-Lys-D-Ala-D-Ala. A series of ligands (Gly, D-Ala, D-Ala-D-Ala and alpha-N-Ac-L-Lys-D-Ala-D-Ala) were immobilized on an N-hydroxysuccinimide-activated agarose support and evaluated using the glycopeptides vancomycin and the aridicin complex. Conditions were developed to enable complete adsorption and efficient elution of both antibiotics. Of the four ligands, the readily available dipeptide offered the best compromise between high binding specificity and recovery on elution. Binding and subsequent high recovery of biologically active products were observed for eight other glycopeptide antibiotics. Column performance was shown by purification of vancomycin directly from a low titer fermentation broth. The applicability of this technique to large scale isolation was demonstrated by the preparative affinity chromatography of 36 g of the aridicins.

Anti-Bacterial Agents↗

PubMed1977

Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.

Acetaldehyde↗

PubMed2004

Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II).

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.

Absorption↗

PubMed1982

LM fibroblast plasma membrane subfractionation by affinity chromatography on con A-sepharose.

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-sided-out orientation of the vesicles. Similarly and asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5'-nucleotidase activity, and (Na+ + K+)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.

Animals↗

PubMed2007

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography.

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.

Chromatography, Affinity↗

PubMed1993

Affinity chromatography to remove viruses during preparation of plasma derivatives.

The use of affinity chromatography methods to purify proteins from complex solutions is well established. The specific use of immuno-affinity chromatography, wherein antibody directed toward a target protein is utilized, has proven to be a particularly effective purification method. Application of immuno-affinity chromatography methods for the isolation of plasma derivatives has now been established and the purity of such products has been truly remarkable. Studies conducted by Baxter have compared the effect of immuno-affinity chromatography on the separation of plasma protein contaminants and model viruses from Factor VIII:C. The results of these studies have indicated that plasma protein contaminant (eg. fibrinogen) reduction during the process of Factor VIII:C immuno-affinity chromatography occurs more gradually than does virus reduction. By using residual plasma protein contamination in Factor VIII:C preparations, the general effectiveness of the immuno-affinity chromatography step in removing virus can be routinely evaluated. Determinations made by Baxter have indicated that greater than 4 logs of reduction in protein contamination is routinely achieved during Factor VIII:C immuno-affinity chromatography at manufacturing scale. Bench scale studies have confirmed this result as well as showing the reduction of model viruses. While the viral safety of plasma derivatives is dependent on the overall manufacturing process, it is clear that immuno-affinity chromatography offers a powerful means of reducing product contamination and enhancing safety.

Biological Products↗

PubMed1985

Purification of cartilage-derived growth factor by heparin affinity chromatography.

Cartilage-derived growth factor (CDGF) was found to bind tightly to columns of immobilized heparin and could be eluted with concentrations of salt in the order of 1.6-1.8 M NaCl. The molecular weight of CDGF was estimated to be 18,000-20,000 by high performance liquid-size exclusion chromatography. The affinity of CDGF for heparin greatly facilitated its purification. Highly purified CDGF active at about 1-2 ng/ml was obtained when crude cartilage extract was applied to heparin-Sepharose and the growth factor activity was recycled over heparin-Sepharose two more times. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain visualization of highly purified CDGF showed one major polypeptide band with a molecular weight of about 19,000 containing over 95% of the protein and one minor polypeptide band containing the rest of the protein. Only the Mr 19,000 polypeptide was active after elution from the polyacrylamide gel. Although CDGF bound tightly to immobilized heparin, it did not bind to immobilized chondroitin sulfate or hyaluronic acid. In addition, CDGF bound to heparin much more tightly than did platelet-derived growth factor even though these two growth factors had similar isoelectric points of about 10. These results suggest that the binding of CDGF to heparin was due to a specific affinity of the 2 molecules for each other.

Animals↗

PubMed1974

Evaluation of equilibrium constants by affinity chromatography.

Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100-lysozyme-d-glucose system in acetate-chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m(-1) and 1.2x10(6)m(-1) are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 10(5) glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.

Acetates↗

PubMed2006

Improved performance of column chromatography by arginine: dye-affinity chromatography.

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.

Arginine↗

PubMed1994

Separation of complexes of major histocompatibility class II molecules and known antigenic peptide by metal chelate affinity chromatography.

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exists for separation of complexes of a known antigenic epitope and MHC class II from empty MHC class II and complexes of MHC class II and endogenously bound peptide. Here we describe an one step metal chelate affinity chromatography method to purify complexes of MHC class II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB1*1501/DRB5*0101) and a peptide analog from human myelin basic protein MBP(84-102) containing a 6 histidine tag (6 x His) and a tyrosine residue at the N-terminus end [6 x His-MBP(83-102)Y83] were prepared and purified. The absence of residual free 6 x His-MBP peptide in the complex preparations were confirmed by gel filtration and TLC analyses. The purified complexes were applied onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose affinity support and 6 x His-tagged peptide class II complexes were selectively eluted with imidazole-containing buffer. The quantitation of bound peptide in the eluted complexes showed 100% occupancy of HLA-DR2 (DRB1*1501/DRB5*0101) with [6 x His-MBP(83-102)Y83] peptide with a recovery of 50-75%. The presence of a single peptide entity in the eluted complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni2+.NTA eluted complexes when analyzed by two-dimensional IEF gel electrophoresis. Finally, we demonstrate that both MBP(84-102) and [6 x His-MBP(83-102)Y83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous antigen presenting cells (APCs). These results demonstrate that metal chelate affinity chromatography can be used to prepare MHC class II-peptide complexes containing single peptide. Such complexes of class II molecules containing known peptide have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptor (TCR).

Amino Acid Sequence↗