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Affinity chromatography

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https://www.readbyqxmd.com/read/28635559/purification-of-antibody-against-ara-h-2-by-a-home-made-immunoaffinity-chromatography-column
#1
Zhihua Wu, Kun Li, Shaode Zhan, Ping Tong, Xin Li, Anshu Yang, Hongbing Chen
Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody (PcaAb), a home-made immunoaffinity chromatography column (IAC) method was established. The properties of the home-made column were compared with those of the Mab Affinity Protein G Agarose High Flow (MPG), a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG...
June 21, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28633910/gentle-antibody-mimetic-affinity-chromatography-with-polyol-responsive-nanoclamps
#2
EDITORIAL
Richard R Burgess, James D Watson
No abstract text is available yet for this article.
June 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28633515/crystal-structure-and-functional-characterization-of-a-cytochrome-p450-bacyp106a2-from-bacillus-sp-pamc-23377
#3
Ki-Hwa Kim, Chang Woo Lee, Bikash Dangi, Sun-Ha Park, Hyun Park, Tae-Jin Oh, Jun Hyuck Lee
Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity (Kd value) to 4-androstenedione was 387 ± 37 µM...
June 21, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28633099/simultaneous-and-specific-enrichment-of-several-amphenicol-antibiotics-residues-in-food-based-on-novel-aptamer-functionalized-magnetic-adsorbents-using-hplc-dad
#4
Shengfeng Huang, Ning Gan, Haibo Liu, You Zhou, Yinji Chen, Yuting Cao
In this work, a novel aptamer functionalized magnetic adsorbent was developed and combined with magnetic dispersive solid phase extraction (MDSPE) for selective enrichment of several amphenicol antibiotics residues (chloramphenicol(CAP), thiamphenicol(TAP) and florphenicol(FF)) in foodstuff then determined by High Performance Liquid Chromatography (HPLC)-Diode array detector(DAD). Firstly, a magnetic silica-coated Fe3O4 microsphere(Fe3O4@SiO2) was synthetized by sol-gel method, then it was functionalized by amino groups through 3-Aminopropyltriethoxysilane (APTES) reagent to form Fe3O4@SiO2-NH2; Thirdly, the amino group on Fe3O4@SiO2-NH2 was transferred to carboxylic group via the succinic anhydride to form Fe3O4@SiO2-COOH...
June 12, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28630013/receptor-mediated-endocytosis-of-vicilin-in-callosobruchus-maculatus-coleoptera-chrysomelidae-larval-midgut-epithelial-cells
#5
Daniele Kunz, Gabriel B Oliveira, Adriana F Uchôa, Richard I Samuels, Maria Lígia R Macedo, Carlos P Silva
The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C...
June 16, 2017: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/28629620/effects-of-inhibiting-vps4-support-a-general-role-for-escrts-in-extracellular-vesicle-biogenesis
#6
Charles E Jackson, Benjamin S Scruggs, Jean E Schaffer, Phyllis I Hanson
Extracellular vesicles (EVs) are proposed to play important roles in intercellular communication. Two classes of EVs can be distinguished based on their intracellular origin. Exosomes are generated within endosomes and released when these fuse with the plasma membrane, whereas ectosomes bud directly from the plasma membrane. Studies of EV function have been hindered by limited understanding of their biogenesis. Components of the endosomal sorting complex required for transport (ESCRT) machinery play essential roles in topologically equivalent processes at both the endosome and the plasma membrane and are consistently recovered in EVs, but whether they are generally required to produce EVs is still debated...
June 16, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28627880/site-specific-fucosylation-analysis-identifies-glycoproteins-associated-with-aggressive-prostate-cancer-cell-lines-using-tandem-affinity-enrichments-of-intact-glycopeptides-followed-by-mass-spectrometry
#7
Jianliang Zhou, Weiming Yang, Yingwei Hu, Naseruddin Hoti, Yang Liu, Punit Shah, Shisheng Sun, David J Clark, Stefani N Thomas, Hui Zhang
Fucosylation (Fuc) of glycoproteins plays an important role in regulating protein function and has been associated with the development of several cancer types including prostate cancer (Pca). Therefore, the research of Fuc glycoproteins has attracted increasing attention recently in analytical field. Herein, a strategy based on lectin affinity enrichments of intact glycopeptides followed by mass spectrometry has been established to evaluate the specificities of various Fuc-binding lectins for glycosite-specific Fuc analysis of non-aggressive (NAG) and aggressive (AG) Pca cell lines...
June 19, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28625911/white-shrimp-litopenaeus-vannamei-recombinant-lactate-dehydrogenase-biochemical-and-kinetic-characterization
#8
Ambar A Fregoso-Peñuñuri, Elisa M Valenzuela-Soto, Ciria G Figueroa-Soto, Alma B Peregrino-Uriarte, Manuel Ochoa-Valdez, Lilia Leyva-Carrillo, Gloria Yepiz-Plascencia
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624494/antibacterial-activity-and-phospholipid-recognition-of-the-recombinant-defensin-j1-1-from-capsicum-genus
#9
Francisco Guillén-Chable, Iván Arenas-Sosa, Ignacio Islas-Flores, Gerardo Corzo, Cynthia Martinez-Liu, Georgina Estrada
The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography...
June 14, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624493/expression-of-the-c-terminal-domain-of-human-apolipoprotein-a-i-using-a-chimeric-apolipoprotein
#10
Daniel E Sallee, James V C Horn, Lukas A Fuentes, Paul M M Weers
Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique a methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification...
June 14, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28624308/efficient-heterologous-expression-functional-characterization-and-molecular-modeling-of-annular-seabream-digestive-phospholipase-a2
#11
Nabil Smichi, Houcemeddine Othman, Neila Achouri, Alexandre Noiriel, Soumaya Triki, Vincent Arondel, Najet Srairi-Abid, Abdelkarim Abousalham, Youssef Gargouri, Nabil Miled, Ahmed Fendri
Here we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2)...
June 14, 2017: Chemistry and Physics of Lipids
https://www.readbyqxmd.com/read/28623696/optimizing-purification-process-of-mim-i-bar-domain-by-introducing-atomic-force-microscope-and-dynamics-simulations
#12
Yue Zhang, Zhichao Lou, Xubo Lin, Qiwei Wang, Meng Cao, Ning Gu
MIM (missing in metastasis) is a member of I-BAR (inverse BAR) domain protein family, which functions as a putative metastasis suppressor. However, methods of gaining high purity MIM-I-BAR protein are barely reported. Here, by optimizing the purification process including changing the conditions of cell lysate and protein elution, we successfully purified MIM protein. The purity of the obtained protein was up to ∼90%. High-resolution atomic force microscope (AFM) provides more visual images, ensuring that we can observe the microenvironment around the target protein, as well as the conformations of the purification products following each purification process...
May 26, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28621608/glycosylation-change-of-alpha-1-acid-glycoprotein-as-a-serum-biomarker-for-hepatocellular-carcinoma-and-cirrhosis
#13
Delin Zhang, Jianzhao Huang, Dan Luo, Xinfu Feng, Yan Liu, Yan Liu
AIM: This research is to explore the glycosylation change of alpha-1-acid glycoprotein (AGP) in hepatocellular carcinoma (HCC), cirrhosis and controls. METHODS: The affinity chromatography and lectin affinity techniques were used to separate and enrich glycosylated AGP, and combined with mass spectrometry to identify and relatively quantify the glycopeptides from AGP. RESULTS: The sialylation and fucosylation of AGP were different among HCC, cirrhosis and controls...
May 2017: Biomarkers in Medicine
https://www.readbyqxmd.com/read/28615107/-preparation-and-characterization-of-mouse-polyclonal-antibody-against-conserved-region-of-human-foxo3
#14
Lei Li, Dan Lyu
Objective To purify the recombinant protein specific to conserved region of forkhead box O3 (FOXO3) and prepare mouse anti-human FOXO3 polyclonal antibody. Methods The DNA fragment (aa290-472) encoding conserved domain of FOXO3 was amplified by PCR, and subsequently cloned into pET28a vector. Following transformation into E.coli BL21, the soluble fusion protein His-FOXO3 was induced by IPTG and purified by Ni-NTA affinity chromatography. The purified protein was used to immunize BALB/c mice to generate polyclonal antibody...
June 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28615092/-purification-of-the-recombinant-com1-and-adaa-of-coxiella-burnetii-and-identification-of-the-antigenicity
#15
Jingxian Liu, Yihong Ji, Zhiyang Shi, Yongjun Jiao
Objective To express and purify two kinds of antigens of Coxiella burnetii (C. burnetii), the main outer membrane protein Com1 and the acute disease antigen A (adaA), in prokaryotic expression system and to validate the two recombinant antigens by mass spectrometry and identify their antigenicity. Methods The gene sequences encoding Com1 and adaA were separately synthesized and constructed into the prokaryotic expression vector pET-20b(+). The constructed vectors were transformed into E.coli BL21(DE3), and the recombinant proteins were induced by IPTG...
June 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28612560/-construction-expression-of-her%C3%AE-lbd-prokaryotic-vector-and-the-activity-of-expressed-protein
#16
Yan-Fang Wang, Ji-Jun Zhao, Zai-Min Tian, Yan-Feng Guo
OBJECTIVES: To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain (hERα-LBD) and to evaluate the estrogen receptor ligand binding activity of the expressed protein. METHODS: hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a...
January 2017: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
https://www.readbyqxmd.com/read/28609623/single-step-enrichment-of-n-glycopeptides-and-phosphopeptides-with-novel-multifunctional-ti4-immobilized-dendritic-polyglycerol-coated-chitosan-nanomaterials
#17
Xiajuan Zou, Jianzheng Jie, Bin Yang
Protein glycosylation and phosphorylation, two of the most important post-translational modifications (PTMs) in the proteome play a vital role in regulating a number of complex biological processes and involvement in a variety of diseases. Comprehensive characterization of the phosphoproteome and glycoproteome requires highly specific and sensitive enrichment methods for purification of phosphopeptides and glycopeptides because many glycoproteins and phosphoproteins naturally occur at low abundances and substoichiometry...
June 13, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28608866/a-small-wolbachia-protein-directly-represses-phage-lytic-cycle-genes-in-candidatus-liberibacter-asiaticus-within-psyllids
#18
Mukesh Jain, Laura A Fleites, Dean W Gabriel
Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium "Candidatus Liberibacter asiaticus" and transmitted by Asian citrus psyllids (Diaphorina citri). Two prophage genomes, SC1 and SC2, integrated in "Ca. Liberibacter asiaticus" strain UF506 were described previously, and very similar prophages are found resident in the majority of "Ca. Liberibacter asiaticus" strains described worldwide. The SC1 lytic cycle is marked by upregulation of prophage late genes, including a functional holin (SC1_gp110); these late genes are activated when "Ca...
May 2017: MSphere
https://www.readbyqxmd.com/read/28608392/volatile-organic-compounds-analysis-by-pulsed-glow-discharge-time-of-flight-mass-spectrometry-as-a-structural-elucidation-tool
#19
M Bouza, J Fandino, N Bordel, R Pereiro, A Sanz-Medel
Pulsed glow discharge (PGD) coupled to time of flight mass spectrometry (TOFMS) has been investigated for volatile organic compounds (VOCs) identification and determination. Optimization of PGD operational conditions (chamber design, applied power, pressure and duty cycle) was performed using acetone and benzene as model compounds. During the different optimizations, molecular, fragment and elemental information were obtained when characteristic GD pulse regions were measured. An exploratory study for several VOCs (lineal hydrocarbons, oxygen-containing compounds and aromatic compounds) revealed the capability of the PGD to provide crucial information to elucidate structures (fragments), molecular ions or even proton affinity nature of the molecules; this last information is a consequence of the enriched proton environment generated along the afterglow region for the ionization chamber used...
June 12, 2017: Journal of Mass Spectrometry: JMS
https://www.readbyqxmd.com/read/28607052/efficient-ultra-high-affinity-chromatography-in-a-one-step-purification-of-complex-proteins
#20
Marina N Vassylyeva, Sergiy Klyuyev, Alexey D Vassylyev, Hunter Wesson, Zhuo Zhang, Matthew B Renfrow, Hengbin Wang, N Patrick Higgins, Louise T Chow, Dmitry G Vassylyev
Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10(-14)-10(-17) M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7)...
June 12, 2017: Proceedings of the National Academy of Sciences of the United States of America
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