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Affinity Column (affinity + column)
Selected AbstractsTemplate Refolding by Use of an Antibody-Coupled Affinity ColumnCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005S. Katoh Abstract To improve the efficiency and throughput, the biointeraction between an antigen and antibody was used for refolding in a packed column, in which an antibody against carbonic anhydrase coupled on a gel support was used as a template ligand. A denatured solution of CAB was mixed with a refolding buffer in a mixing chamber, and was supplied to the antibody-coupled column for refolding. Higher refolding efficiencies were obtained in the column than by the batch dilution method at relatively low concentrations of denaturant. By increasing the adsorption capacity of the column, the efficiency of refolding, as well as the throughput, could be increased. [source] Comparison of anti-inflammatory activities of ruscogenin, a major steroidal sapogenin from Radix Ophiopogon japonicus, and Its succinylated derivative, RUS-2HSDRUG DEVELOPMENT RESEARCH, Issue 4 2008Ya-Lin Huang Abstract Ruscogenin (RUS), first isolated from Ruscus aculeatus, is also a major steroidal sapogenin of the traditional Chinese herb Radix Ophiopogon japonicus. It has robust anti-inflammatory activities. In previous studies, a ruscogenin affinity column, derived from succinylated ruscogenin (RUS-2HS), was used to purify an antibody of ruscogenin. A ruscogenin affinity column can also be used to explore its protein targets. However, until now there have been no related pharmacological reports about ruscogenin derivatives. Whether the activity groups of ruscogenin have been blocked during the derivation process remains unknown. The present study was performed to compare the anti-inflammatory activities in vitro of RUS-2HS and ruscogenin. Both compounds reduced tumor necrosis factor-, (TNF-,)-induced adhesion of human pro-myelocytic leukemia cells (HL-60) to endothelial ECV304 cells with IC50 values of 6.90,nM and 7.45,nM, respectively. They were also inhibited overexpression of ICAM-1 in ECV304 cells at the mRNA level as evaluated by real-time PCR and at the protein level evaluated by flow cytometry with similar potency. Such data demonstrate that the functional groups of ruscogenin were not blocked by derivation, suggesting further use of the ruscogenin affinity column for target investigation. Meanwhile, RUS-2HS was found to have remarkable anti-inflammatory activity for the first time, indicating it would be a new lead compound with improved bioavailability. Drug Dev Res 69: 196,202, 2008. © 2008 Wiley-Liss, Inc. [source] Surface grafting of glycidyl methacrylate on silica gel and polyethylene beadsELECTROPHORESIS, Issue 18 2003Seong-Ho Choi Abstract Surface grafting of glycidyl methacrylate (GMA) on silica gel and a polyethylene bead was performed by radical polymerization and radiation-induced polymerization, respectively, in order to improve softness. Subsequently, diethylene triamine (DETA), triethylene tetraamine (TETA), and iminodiacetic acid (IDA) were introduced to the grafted GMA for use as affinity columns. The efficiency of the affinity column was investigated by use of bovine serum albumin (BSA) and hemoglobin (Hb) as model proteins. The affinity degree of BSA was higher than Hb for the DETA and TETA column, whereas the affinity degree of Hb was higher than BSA for the IDA column supported by silica gel. The affinity degree of BSA was higher than Hb for the DETA and TTA column supported by polyethylene (PE) beads. [source] Novel brain 14-3-3 interacting proteins involved in neurodegenerative diseaseFEBS JOURNAL, Issue 16 2005Shaun Mackie We isolated two novel 14-3-3 binding proteins using 14-3-3 , as bait in a yeast two-hybrid screen of a human brain cDNA library. One of these encoded the C-terminus of a neural specific armadillo-repeat protein, ,-catenin (neural plakophilin-related arm-repeat protein or neurojungin). ,-Catenin from brain lysates was retained on a 14-3-3 affinity column. Mutation of serine 1072 in the human protein and serine 1094 in the equivalent site in the mouse homologue (in a consensus binding motif for 14-3-3) abolished 14-3-3 binding to ,-catenin in vitro and in transfected cells. ,-catenin binds to presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer's disease. The other clone was identified as the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). Human IRSp53 interacts with the gene product implicated in dentatorubral-pallidoluysian atrophy, an autosomal recessive disorder associated with glutamine repeat expansion of atrophin-1. [source] Immune Response to a 26-kDa Protein, Alkyl Hydroperoxide Reductase, in Helicobacter pylori-Infected Mongolian Gerbil ModelHELICOBACTER, Issue 4 2001Jing Yan ABSTRACT Background. The host immune response is thought to play an important role in the outcome of Helico-bacter pylori infection. The successful development of the H. pylori -infected Mongolian gerbil model that mimics human disease has enabled study of the antibody response against H. pylori antigens. Materials and Methods. Serum samples from ulcer and carcinogenesis models of H. pylori -infected gerbils were used to screen for H. pylori antigens that cause a humoral immune response in the infected hosts. H. pylori alkyl hydroperoxide reductase (AhpC) is one such antigen on which we report here. The tsaA gene encoding AhpC was amplified by PCR from H. pylori ATCC 43504 strain, cloned into pMALTM -c2 expression vector and expressed in Escherichia coli. Maltose-binding protein fusion protein (MBP-AhpC) was purified by a MBP affinity column. Using purified recombinant AhpC protein as an antigen, the antibody response and changes of antibody levels against AhpC in the gerbil models were studied by Western blotting and ELISA. Results. Antibody against AhpC was negative in the early stages of infection, and became positive in the gerbils with the emergence of gastric diseases such as chronic active gastritis, gastric ulcer and gastric cancer. The antibody levels (ELISA) increased gradually over time and were higher in gerbils with gastric ulcer than that in gerbils without ulcers. Conclusions. Use of the gerbil model that mimics human H. pylori infection is likely to provide insights into the role of H. pylori -specific antigens possibly related to the subsequent development of gastric diseases. [source] A Mu-class glutathione S -transferase from gills of the marine shrimp Litopenaeus vannamei: Purification and characterizationJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2007Carmen A. Contreras-Vergara Abstract Glutathione S -transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S -transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione,agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of ,25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 ,mol/(min mg), and the Km values for CDNB and GSH are very similar (390 and 335 ,M, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver,Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:62,67, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20162 [source] Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolinJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2002Tuula Stark Abstract In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111,118; L. Staffas, L. Mankowitz, M. Söderström, A. Blanck, I. Porsch-Hällström, C. Sundberg, B. Mannervik, B. Olin, J. Rydström, and J.W. DePierre, Biochem J, 1992, 286, 65,72) isoenzymes of GST, primarily of the , class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45,50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:169,173, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10034 [source] PROPERTIES OF CYSTEINE PROTEINASE INHIBITORS FROM BLACK GRAM AND RICE BEANJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2001SOOTTAWAT BENJAKUL ABSTRACT Cysteine proteinase inhibitors (CPI) were purified to 59 and 54 fold from black gram (Vignaraungo (L.) Hepper) and rice bean (Vignaumbellata Thunb.), respectively, by using heal treatment, followed by chromatography on a carboxymethyl (CM)-papain-Sepharose affinity column. The purified inhibitors were highly inhibitory to papain and Pacific whiting cathepsin L in a concentration dependent manner. They were detected as a dark band on tricine-SDS-PAGE gel stained for inhibitory activity. The apparent molecular weights of purified CPI from black gram and rice bean seeds were estimated to be 12, 000 daltons. The purified inhibitors were thermostable up to 90C and active in the neutral and alkaline pH ranges. [source] Glucose-regulated protein 78: A new partner of p53 in trophoblastPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2009Serge Arnaudeau Abstract Although wild-type p53 protein is overexpressed in first trimester trophoblast, it is inactive towards its target genes Metalloproteinase 2 and 9. This seems to be due to a complex mechanism of inactivation and stabilization of p53 relying on the formation of protein complexes involving the N-terminus of p53. To detect the proteins associated with this sequence, we incubated biotinylated p53 N-terminal peptide in cytotrophoblastic cell medium 24,h before lysis of cells. We purified the proteins retained on biotinylated peptide using a neutravidin affinity column. Proteins were then identified by peptide mass finger printing followed or not by peptide fragmentation sequencing. Among these proteins, we identified glucose-regulated protein 78 (GRP78) and verified its interaction with p53 in trophoblastic cells by immunoprecipitation and Western blot analysis. Moreover, the decreased expression of GRP78 induced by GRP78siRNA or versipelostatin decreased the formation of high molecular weight p53 complexes and p53 monomer and increased trophoblastic invasion. These results suggest that GRP78 is involved in inactivation and stabilization of p53 and in the regulation of trophoblastic invasion. [source] Placental Trophoblast from Successful Human Pregnancies Expresses the Tolerance Signaling Molecule, CD200 (OX-2),AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2003David A. Clark Problem: Th1 cytokine-dependent abortions in the CBA × DBA/2 mouse model have been linked to down-regulation of expression of the CD200 (OX-2) ,tolerance' signal on trophoblast and in decidua prior to onset of the abortion process. Abortions could be prevented by administration of a soluble CD200. Is CD200 expressed on trophoblast in successful human pregnancy? Method of study: As one cannot easily obtain trophoblasts in large quantities from successful human pregnancies in the first trimester prior to the onset of the abortion process at 6 weeks gestation, we examined as a first step, trophoblast isolated from term placentae (i.e. successful pregnancies). CD9, trophoblasts were isolated by affinity column and stained for intracellular cytokeratin, and surface CD200 using PE-anti-human CD200 monoclonal antibody. mRNA was extracted from CD9+ and CD9, cells and tested by reverse transcription,polymerase chain reaction for CD200 mRNA. CD9, placental cells were separated by velocity sedimentation and test for CD200-dependent suppression of an allogeneic human mixed lymphocyte culture where cytotoxic T cell (CTL) generation, and Th1 , Th2 cytokine production shift were measured. Results: CD9, but not CD9+ placental cell populations contained cells with mRNA for CD200, both a normal length transcript and a truncated transcript. Flow cytometry showed a CD200+ cytokeratin+ moderate-to-large-sized cell population compatible with trophoblasts and a smaller subset of cytokeratin, cells that expressed CD200 at normal and at high levels. The moderate-sized population proved most potent at inhibiting CTL generation and caused a Th1,Th2 cytokine shift. These effects were blocked by monoclonal anti-CD200. Conclusions: A subpopulation of cytokeratin+ placental trophoblasts express bioactive CD200 able to alter maternal immune responses in a favorable (Th2 > Th1) direction. Two populations of CD200+ small- and medium-small-sized cytokeratin, placental cells remain to be identified. Studies of karyotyped first trimester elective termination and spontaneous miscarriage tissues are needed. [source] Glutathione S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphidARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2005Frédéric Francis Abstract Glutathione S-transferases (GST) in insects play an important role in the detoxification of many substances including allelochemicals from plants. Induction of GST activity in Myzus persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and confirmed using artificial diet with pure allelochemicals added. The 2,4-dinitro-1-iodobenzene (DNIB) was found to be a useful substrate for identifying particular GSTs in insects. GSTs from M. persicae were purified using different affinity chromatography columns and related kinetic parameters were calculated. GST isoenzymes were characterised using electrophoretic methods. Although SDS-PAGE results indicated similarity among the purified enzymes from each affinity column, biochemical studies indicated significant differences in kinetic parameters. Finally, the GST pattern of M. persicae was discussed in terms of insect adaptation to the presence of plant secondary substances such as the glucosinolates and the isothiocyanates, from Brassicaceae host plants. Arch. Insect Biochem. Physiol. 58:166,174, 2005. © 2005 Wiley-Liss, Inc. [source] Anti,citrullinated protein antibodies bind surface-expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor , productionARTHRITIS & RHEUMATISM, Issue 5 2010Ming-Chi Lu Objective Anti,citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA. Methods ACPAs were purified from pooled ACPA-positive RA sera by cyclic citrullinated peptide,conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor , (TNF,) production and NF-,B DNA binding activity of the cells were measured by enzyme-linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis. Results ACPAs specifically enhanced TNF, production and increased the DNA-binding activity of NF-,B in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface,expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S-transferase,tagged recombinant Grp78 (97.52 kd) became a 72-kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNF, production. Conclusion We clearly demonstrated that ACPAs enhance NF-,B activity and TNF, production in monocyte/macrophages via binding to surface-expressed citrullinated Grp78. [source] Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Lei Jin NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0,Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2,Å resolution and a clear molecular-replacement solution was obtained for solution of the structure. [source] Limitation of immunoaffinity column for the removal of abundant proteins from plasma in quantitative plasma proteomicsBIOMEDICAL CHROMATOGRAPHY, Issue 5 2009Tomoko Ichibangase Abstract In plasma proteomics, before a proteome analysis, it is essential to prepare protein samples without high-abundance proteins, including albumin, via specific preparation techniques, such as immunoaffinity capture. However, our preliminary experiments suggested that functional changes with use alter the ability of the immunoaffinity column. Thus, in this study, to evaluate the changes of the removal ability of abundant proteins from plasma by the immunoaffinity column, plasma proteome analysis was performed for the long-term test for the reproducibility of the affinity column using the fluorogenic derivatization,liquid chromatography,tandem mass spectrometry method combined with an IgY column. The specific adsorption for albumin decreased with an increase in the number of the column usage before its expiration date. Moreover, it was demonstrated that hydrophobic high molecular weight compounds in plasma adsorbed onto the column materials surface contributed to the functional changes from specific immunoaffinity adsorption into hydrophobic interaction. These results suggested that, in quantitative plasma proteomics studies, it is important to keep in mind the risk of not only the nonselective loss but also the changes in the adsorption ability of the immunoafinity column. Copyright © 2008 John Wiley & Sons, Ltd. [source] High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolithBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Quanzhou Luo Abstract Reactive monoliths of macroporous poly(glycidyl methacrylate- co -ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 481,489, 2002. [source] Affinity Ligand Selection from a Library of Small Molecules: Assay Development, Screening, and ApplicationBIOTECHNOLOGY PROGRESS, Issue 1 2005Lakshmi D. Saraswat A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work. [source] Surface grafting of glycidyl methacrylate on silica gel and polyethylene beadsELECTROPHORESIS, Issue 18 2003Seong-Ho Choi Abstract Surface grafting of glycidyl methacrylate (GMA) on silica gel and a polyethylene bead was performed by radical polymerization and radiation-induced polymerization, respectively, in order to improve softness. Subsequently, diethylene triamine (DETA), triethylene tetraamine (TETA), and iminodiacetic acid (IDA) were introduced to the grafted GMA for use as affinity columns. The efficiency of the affinity column was investigated by use of bovine serum albumin (BSA) and hemoglobin (Hb) as model proteins. The affinity degree of BSA was higher than Hb for the DETA and TETA column, whereas the affinity degree of Hb was higher than BSA for the IDA column supported by silica gel. The affinity degree of BSA was higher than Hb for the DETA and TTA column supported by polyethylene (PE) beads. [source] Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptidesFEBS JOURNAL, Issue 20 2000Jocelyne Guay The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S -transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S -transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6,6.3 nm for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nm) and cathepsin L (Ki = 0.12 nm) compared with cathepsin S (Ki = 65 nm). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nm) than cathepsin S (Ki = 7.6 nm) itself or cathepsin K (Ki = 7.0 nm). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J.E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745,750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained. [source] Immunodetection and Characterization of Antigens Expressed by Uncinula necatorJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2002V. L. Markovic Abstract Conidia from four genetically distinct isolates of Uncinula necator (Schw.) Burr. were used to raise a polyclonal antiserum. Immunofluorescent detection of the fungus on Vitis vinifera (cv. Chardonnay) indicated that the antiserum bound specifically to fungal antigens present on both conidia and hyphae, with no detection of underlying berry tissues. The antibody reacted with three antigens present on the conidia (Mr 21, 29 and > 250 kDa). Immunoreactivity with the 21 kDa antigen was dependent upon the preservation of at least one disulphide bond linkage. Evidence from immunoblot staining and enzyme immunoassay indicated that only a small proportion of the recognized epitopes contained carbohydrate moieties. The antibody detected homologous U. necator conidial antigens in a plate trapped antigen-enzyme-linked immunoabsorbent assay (PTA-ELISA), with a linear range of detection extending from 1000 to 9000 conidia/ml at a 1/5000 dilution of serum. Under these conditions the immunoassay also detected antigens from pooled heterologous U. necator isolates. The antiserum exhibited cross-reactivity with antigens present on Aspergillus, Pithomyces and Sporobolomyces species co-isolated from powdery mildew-infected grapes, which could not be removed by fractionation of the antiserum on antigen affinity columns. Monoclonal antibodies were subsequently produced to avoid the problems of cross-reactivity associated with the polyclonal antibody. Antibodies produced from two clonal lines exhibited specificity for U. necator and were shown to detect a 21 kDa conidial antigen. Use of either of these antibodies enabled the differentiation of grapes grouped on the basis of powdery mildew disease levels. [source] Detection of the human GPR50 orphan seven transmembrane protein by polyclonal antibodies mapping different epitopesJOURNAL OF PINEAL RESEARCH, Issue 1 2007Hassina Ould Hamouda Abstract:, GPR50 is an orphan seven transmembrane protein related to the melatonin receptor subfamily comprising MT1 and MT2 receptors. In the absence of any known ligand for GPR50, other tools are critical for the characterization of this protein. Here, we describe the generation, purification and characterization of the first rabbit polyclonal antibodies generated against peptides corresponding to the N-terminus, C-terminus and two additional regions within the intracellular tail of GPR50. Immune sera were purified on peptide-antigen affinity columns. Antibodies specifically recognized a GPR50-YFP fusion protein on the plasma membrane of HEK 293 cells in immunofluorescence experiments. In Western blot experiments, the monomeric and dimeric forms of GPR50 were detected as proteins of 66 and 130 kDa, respectively. In addition, these new antibodies were sufficiently sensitive to detect GPR50 in brain slices of the rat pituitary and human hippocampus. In conclusion, we successfully produced antibodies against the orphan GPR50 protein that will become valuable tools for functional studies of this protein. [source] Kinetic studies of biological interactions by affinity chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2009John E. Schiel Abstract The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This paper will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this paper and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. [source] High-throughput quantification of selenium in individual serum proteins from a healthy human population using HPLC on-line with isotope dilution inductively coupled plasma-MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2010Sophia Letsiou Abstract In this study, a method, based on dual column affinity chromatography hyphenated to isotope dilution inductively coupled plasma,quadrupole MS, was developed for selenium determination in selenoprotein P, glutathione peroxidase, and selenoalbumin in human serum samples from a group of healthy volunteers (n=399). Method improvement was achieved using methanol-enhanced isotope dilution which resulted in improved sensitivity and removal of isobaric interferences. Although no human serum reference materials are currently certified for their selenium species levels, method development was conducted using human serum reference material BCR 637 and 639 as their Se species content has been reported in the previous studies, and thus comparisons were possible. The mean selenium concentrations determined for the 399 healthy volunteer serum samples were 23±10,ng Se mL,1 for glutathione peroxidase, 49±15,ng Se mL,1 for selenoprotein P and 11±4,ng Se mL,1 for selenoalbumin. These values are found to be in close agreement with published values for a limited number of healthy volunteer samples, and to establish baseline Se levels in serum proteins for an apparently healthy group of individuals, thus allowing for subsequent comparisons with respective values determined for groups of individuals with selenium related health issues, as well as assist in the discovery of potential selenium biomarkers. Also, the relationship between Se serum protein levels and some anthropometric characteristics of the volunteer population were investigated. Additionally, further development of the analytical method used in this study was achieved by adding a size exclusion chromatography column after the two affinity columns via a switching valve. This allowed for the separation of small selenium-containing molecules from glutathione peroxidase and thus enhanced the overall confidence in its identification. [source] Crystallization and preliminary X-ray crystallographic analysis of p24, a component of the potato nuclear factor PBF-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2002Darrell Desveaux The Solanum tuberosum (potato) nuclear factor PBF-2 is implicated in pathogen-induced expression of the pathogenesis-related gene PR-10a. Crystals of the DNA-binding component of PBF-2, p24, have been obtained at 277,K in 20,mM Tris,HCl pH 8.0. Recombinant protein with a His tag at its C-terminus was overexpressed in Escherichia coli in the presence and absence of selenomethionine and was purified using a combination of HiTrap affinity columns and gel-filtration chromatography. Crystals suitable for structural analysis were obtained for both native and selenomethionine-labelled proteins and yielded diffraction data at 100,K that were processed to 2.3 and 2.8,Å resolution, respectively. The p24 protein crystals belong to space group P212121, with unit-cell parameters a = 69.4,(69.1), b = 89.4,(90.5), c = 144.1,(144.3),Å. The asymmetric unit contains four protomers, giving a crystal volume per protein mass (VM) of 2.23,Å3,Da,1 and a solvent content of 45% by volume. [source] High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolithBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Quanzhou Luo Abstract Reactive monoliths of macroporous poly(glycidyl methacrylate- co -ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 481,489, 2002. [source] Differential phosphoproteome profiling reveals a functional role for VASP in Helicobacter pylori -induced cytoskeleton turnover in gastric epithelial cellsCELLULAR MICROBIOLOGY, Issue 11 2008Olivia Knauer Summary Infection with Helicobacter pylori induces various gastric diseases, including ulceration, gastritis and neoplasia. As H. pylori -induced cellular mechanisms leading to these disease states are widely unclear, we analysed the phosphoproteome of H. pylori -infected gastric epithelial cells. Phosphoproteins from infected cells were enriched using affinity columns and analysed by two-dimensional gel electrophoresis and mass spectrometry. Eleven novel phosphoproteins that showed differentially regulated phosphorylation levels during H. pylori infection were identified. Interestingly, the identified proteins were actin-binding, transport and folding, RNA/DNA-binding or cancer-associated proteins. We analysed functions of one identified H. pylori -regulated candidate, the vasodilator-stimulated phosphoprotein (VASP). H. pylori induced VASP phosphorylation at residues Ser157, Ser239 and Thr278, which was enhanced by the bacterial oncogene cytotoxin-associated gene A. Overexpression of a phosphorylation-resistant VASP mutant efficiently blocked host cell elongation. We identified cGMP-dependent protein kinase G-mediated Ser239 and Thr278 phosphorylation of VASP as a crucial event in H. pylori -dependent host cell elongation. These results suggest that phosphorylated VASP could be a novel target candidate for therapeutic intervention in H. pylori -related gastric diseases. [source] | ||||||||||||||||