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Affinity chromatography

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https://www.readbyqxmd.com/read/28731574/rapid-buffer-and-ligand-screening-for-affinity-chromatography-by-multiplexed-surface-plasmon-resonance-imaging
#1
Karin P M Geuijen, Daniëlle E J W van Wijk-Basten, David F Egging, Richard B M Schasfoort, Michel H Eppink
Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g. microtiterplates, robocolumns). We have studied the application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process...
July 21, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28728084/purification-of-monoclonal-antibodies-entirely-in-flow-through-mode
#2
Tsuyoshi Yamada, Koichi Yamamoto, Takashi Ishihara, Shigeru Ohta
A depth filter is widely used in biopharmaceutical manufacturing process. In this study, we found that buffer exchange to reduce conductivity dramatically improved the removal of impurities in depth filtration. The host cell protein clearance was comparable to that of protein A affinity chromatography, which is generally used as a first capture step in monoclonal antibody purification. In addition, a combination of different depth filters showed enhanced purification. Additional flow-through purification is possible without any sample preparation, and a biopharmaceutical-quality purity level (<100ppm for host cell protein and <5% for high molecular weight species) can be attained...
July 3, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28724286/deep-dive-targeted-quantification-for-ultrasensitive-analysis-of-proteins-in-non-depleted-human-blood-plasma-serum-and-tissues
#3
Song Nie, Tujin Shi, Thomas L Fillmore, Athena A Schepmoes, Heather Brewer, Yuqian Gao, Ehwang Song, Hui Wang, Karin D Rodland, Wei-Jun Qian, Richard D Smith, Tao Liu
Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in non-depleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundance proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging, especially for these samples without available antibodies for enrichment...
July 20, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28722797/highly-selective-enrichment-of-phosphopeptides-by-on-chip-indium-oxide-functionalized-magnetic-nanoparticles-coupled-with-maldi-tof-ms
#4
Dandan Jiang, Naizhong Song, Xiqian Li, Jiutong Ma, Qiong Jia
Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein-related samples prior to MS-based analysis. Herein, an on-chip system coupled magnetic SPE with MALDI-TOF MS was designed. A metal oxide affinity chromatography (MOAC) material, indium oxide, was coated on the surface of Fe3 O4 magnetic nanoparticles (MNPs) to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β-casein and BSA (1:1000) and the mixture of β-casein, BSA, and ovalbumin (1:100:100)...
July 19, 2017: Proteomics
https://www.readbyqxmd.com/read/28716760/properties-of-purified-cyp2r1-in-a-reconstituted-membrane-environment-and-its-25-hydroxylation-of-20-hydroxyvitamin-d3
#5
Chloe Y S Cheng, Tae-Kang Kim, Saowanee Jeayeng, Andrzej T Slominski, Robert C Tuckey
Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase...
July 14, 2017: Journal of Steroid Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28716424/molecular-cloning-of-an-ester-forming-triterpenoid-udp-glucose-28-o-glucosyltransferase-involved-in-saponin-biosynthesis-from-the-medicinal-plant-centella-asiatica
#6
Fernanda de Costa, Carla J S Barber, Yeon-Bok Kim, Darwin W Reed, Haixia Zhang, Arthur G Fett-Neto, Patrick S Covello
Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified...
September 2017: Plant Science: An International Journal of Experimental Plant Biology
https://www.readbyqxmd.com/read/28715685/metabolic-profile-study-of-7-8-dihydroxyflavone-in-monkey-plasma-using-high-performance-liquid-chromatography-tandem-mass-spectrometry
#7
Taoping Sun, Sijing Chen, Hao Huang, Tianqing Li, Wei Yang, Liegang Liu
7, 8-Dihydroxyflavone, as a high-affinity tropomyosin-receptor-kinase B agonist, can mimic the physiological actions of brain-derived neurotrophic factor and exert a variety of neurological actions in numerous models including Parkinsońs disease, depression, learning and memory. Nonetheless, a limited number of studies have been focused on its metabolism in mammal and no methodology has been reported for the determination of 7, 8-DHF and its metabolites. Herein, we developed a rapid, sensitive and accurate method using high performance liquid chromatography-tandem mass spectroscopy for the determination of 7, 8-DHF and its metabolites in monkey plasma...
July 4, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28715189/diamine-tethered-bis-thiourea-organocatalyst-for-asymmetric-henry-reaction
#8
Jan Otevrel, Pavel Bobal
We have developed a novel multifunctional C2-symmetric biphenyl-based diamine-tethered bis(thiourea) organocatalyst, which was tested in the asymmetric Henry reaction. Under thoroughly optimized conditions, the catalyst provided exceptionally high yields and excellent enantioselectivities especially for electron-deficient aromatic and heterocyclic substrates. Due to high affinity of the catalyst to silica gel, a simple chromatography-free nitroaldol isolation procedure was feasible. Preliminary kinetic and spectroscopic experiments were performed in order to complete the mechanistic picture of the organocatalyzed nitroaldolisation process...
July 17, 2017: Journal of Organic Chemistry
https://www.readbyqxmd.com/read/28713775/salivary-tick-cystatin-omc2-targets-lysosomal-cathepsins-s-and-c-in-human-dendritic-cells
#9
Tina Zavašnik-Bergant, Robert Vidmar, Andreja Sekirnik, Marko Fonović, Jiří Salát, Lenka Grunclová, Petr Kopáček, Boris Turk
To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28712956/refolding-of-a-novel-cholesterol-oxidase-from-pimelobacter-simplex-reveals-dehydrogenation-activity
#10
Hui-Min Qin, Jian-Wen Wang, Qianqian Guo, Songtao Li, Panpan Xu, Zhangliang Zhu, Dengyue Sun, Fuping Lu
Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coli BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28711789/towards-a-better-understanding-of-protein-changes-in-common-bean-under-drought-a-case-study-of-n-glycoproteins
#11
Tanja Zadražnik, Anders Moen, Wolfgang Egge-Jacobsen, Vladimir Meglič, Jelka Šuštar-Vozlič
Drought is one of the major abiotic stress conditions limiting crop growth and productivity. Glycosylation of proteins is very important post-translational modification that is involved in many physiological functions and biological pathways. To understand the involvement of N-glycoproteins in the mechanism of drought response in leaves of common bean, a proteomic approach using lectin affinity chromatography, SDS-PAGE and LC-MS/MS was applied. Quantification of N-glycoproteins was performed using MaxQuant with a label free quantification approach...
July 5, 2017: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/28711733/expression-and-purification-of-tat-ndrg2-recombinant-protein-and-evaluation-of-its-anti-proliferative-effect-on-lncap-cell-line
#12
Fahimeh Farokhinejad, Abbas Behzad Behbahani, Gholam Reza Rafiei Dehbidi, Mohammad Ali Takhshid
N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E...
July 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28710796/plant-produced-zika-virus-envelope-protein-elicits-neutralizing-immune-responses-that-correlate-with-protective-immunity-against-zika-virus-in-mice
#13
Ming Yang, Haiyan Sun, Huafang Lai, Jonathan Hurtado, Qiang Chen
The global Zika virus (ZIKV) outbreak and its link to fetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant-produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes...
July 15, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28710679/new-phage-display-isolated-heptapeptide-recognizing-the-regulatory-carboxy-terminal-domain-of-human-tumour-protein-p53
#14
Sihem Ben Abid, Mouna Sahnoun, Ines Yacoubi-Hadj Amor, Salma Abdelmoula-Souissi, Hajer Hassairi, Raja Mokdad-Gargouri, Ali Gargouri
The transcription factor tumor protein p53 (P53) controls a variety of genes most involved in cell cycle and is at the origin of apoptosis when DNA is irreparably damaged. We planned to select novel tumor protein p53-interacting peptides through the screening of hepta-peptide phage-display libraries. For this aim, human tumor suppressor protein p53 was expressed in Escherichia coli as Glutathione S-transferase fusion and purified by affinity chromatography. The phage library was then screened on this immobilized protein target...
July 14, 2017: Protein Journal
https://www.readbyqxmd.com/read/28709863/dissociation-and-purification-of-the-endogenous-membrane-bound-vo-complex-from-pichia-pastoris
#15
Sumei Li, Tao Hong, Kun Wang, Yinghong Lu, Min Zhou
Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification...
July 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28708533/expression-and-molecular-cloning-of-interferon-stimulated-genes-in-buffalo-bubalus-bubalis
#16
Nipuna Thakur, Girjesh Singh, A Paul, J Bharati, G Rajesh, Vidyalakshmi Gm, V S Chouhan, S K Bhure, V P Maurya, G Singh, M Sarkar
Buffalo, the most important livestock species in tropical India, remains to be a poor breeder mainly due to embryonic mortality (65%) occurring mostly between 16 and 18 days of pregnancy. Early and accurate diagnosis of pregnancy can thus become a boon for successful herd management in buffalo. However, most of the currently available methods allow diagnosis only after 30 days post AI. Interferon tau (IFNT), the first pregnancy recognition signal in ruminants is one such molecule, which stimulates expression of various Interferon stimulated genes (ISGs) in the peripheral blood mononuclear cells (PBMC's) concomitant with IFNT signaling which occurs around maternal recognition of pregnancy (MRP)...
June 1, 2017: Theriogenology
https://www.readbyqxmd.com/read/28707740/design-of-a-ruthenium-iii-immobilized-affinity-material-based-on-a-%C3%AE-cyclodextrin-functionalized-poly-glycidyl-methacrylate-ethylene-dimethacrylate-monolith-for-the-enrichment-of-hippuric-acid
#17
Xuemei Li, Xiqian Li, Haijiao Zheng, Jiutong Ma, Guoxing Xu, Qiong Jia
Immobilized metal affinity chromatography has drawn great attention as a widespread separation and purification approach. In this work, ruthenium was firstly introduced into the preparation of immobilized metal affinity chromatography considering its affinity to N,O-donor ligands. A β-cyclodextrin-functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) monolith was designed and employed as the supporting material in immobilized metal affinity chromatography. Thiosemicarbazide was introduced into the synthesis process, which not only acted as a bridge between β-cyclodextrin and glycidyl methacrylate, but also chelated with ruthenium because of its mixed hard-soft donor characteristics...
July 14, 2017: Journal of Separation Science
https://www.readbyqxmd.com/read/28707490/transferon%C3%A2-a-peptide-mixture-with-immunomodulatory-properties-is-not-immunogenic-when-administered-with-various-adjuvants
#18
Sandra Avila, Leslie Muñoz-García, Said Vázquez-Leyva, Nohemí Salinas-Jazmín, Emilio Medina-Rivero, Lenin Pavón, Gabriela Mellado-Sánchez, Rommel Chacón-Salinas, Sergio Estrada-Parra, Luis Vallejo-Castillo, Sonia Mayra Pérez-Tapia
Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (< 10 kDa) and is used to treat diseases with an inflammatory component. Some biotherapeutics, including those composed of peptides, can induce anti-drug antibodies (ADA) that block or diminish their therapeutic effect. Nevertheless, few studies have evaluated peptide-derived drug immunogenicity. In this study, the immunogenicity of Transferon was examined in a murine model during an immunization scheme using the following adjuvants: Al(OH)3, incomplete Freund's adjuvant (IFA), or Titermax Gold...
December 2017: Journal of Immunotoxicology
https://www.readbyqxmd.com/read/28706183/modulation-of-interleukin-12-activity-in-the-presence-of-heparin
#19
Srinivas Jayanthi, Bhanu Prasanth Koppolu, Khue G Nguyen, Sean G Smith, Barbara K Felber, Thallapuranam Krishnaswamy Suresh Kumar, David A Zaharoff
Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold. Conversely, other GAGs did not demonstrate significant binding, nor did their addition affect hIL-12 bioactivity...
July 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28704719/biochemical-and-functional-analysis-of-corticotropin-releasing-factor-purified-from-an-aqueous-extract-of-human-placenta-used-as-wound-healer
#20
Namrata Singh, Debasish Bhattacharyya
Human placental extract constitutes of innumerable therapeutically important components mostly used in wound healing arising from the skin and burn injuries. However, there is still some bioactive present in the placental extracts yet to be characterized to better under the complex process of wound healing mediated by the placental extract. In this study, the presence of corticotropin releasing factor (CRF) in an aqueous extract of human placenta was detected and quantified by dot blot and CRF-ELISA immunoassay kit respectively...
June 28, 2017: Journal of Pharmaceutical and Biomedical Analysis
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