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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/28634775/hydrophilic-acylated-surface-protein-a-haspa-of-leishmania-donovani-expression-purification-and-biophysico-chemical-characterization
#2
Manoj Kumar, Kishu Ranjan, Vijay Singh, Chandramani Pathak, Anju Pappachan, Desh Deepak Singh
Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a "non-classical" pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils...
June 21, 2017: Protein Journal
https://www.readbyqxmd.com/read/28634114/peanut-digestome-identification-of-digestion-resistant-ige-binding-peptides
#3
Luigia Di Stasio, Gianluca Picariello, Mariantonietta Mongiello, Rita Nocerino, Roberto Berni Canani, Simona Bavaro, Linda Monaci, Pasquale Ferranti, Gianfranco Mamone
Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases...
June 17, 2017: Food and Chemical Toxicology
https://www.readbyqxmd.com/read/28633930/phospholipase-a2-in-the-venom-of-three-cottonmouth-snake-species
#4
Ying Jia, Boris Ermolinsky, Aryana Garza, Daniele Provenzano
Two phospholipase A2s (PLA2s), Asp49 (D49) and Lys49 (K49), were purified by one-step reverse-phase high performance liquid chromatography (RP-HPLC) from the venom of each of the three subspecies of cottonmouth snake, Western cottonmouth (Agkistrodon piscivorus leucostoma; Apl), Eastern cottonmouth (Agkistrodon piscivorus piscivorus; App) and Florida cottonmouth (Agkistrodon piscivorus conanti; Apc). Venom protein profiles and PLA2s elution pattern of the three cottonmouth snakes were remarkably similar displaying four similar sharp and two wide peaks; in all cases K49 PLA2 eluted first followed by D49 PLA2...
June 17, 2017: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/28631997/expression-and-purification-of-a-functional-porcine-soluble-triggering-receptor-expressed-on-myeloid-cells-1
#5
Hui Li, Shuangshuang Guo, Leyan Yan, Chunhua Meng, Yiyi Hu, Kongwang He, Zhendan Shi
Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a vital role in the pathogen-triggered amplification loop required for proinflammatory responses. Blockade of TREM-1 signaling may inhibit expansion of sepsis and prolong survival of animals. In the present study, the gene of porcine soluble TREM-1 was cloned and expressed in E. coli. After purification, the bioactivity of recombinant porcine soluble TREM-1 was tested in vitro on porcine alveolar macrophages. The results showed that supplementation with the recombinant porcine sTREM-1 protein rapidly and dose-dependently attenuated the upregulation of cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12, IL-16, IL-18, and TNF-α) caused by LPS stimulation in the cultured porcine alveolar macrophages...
February 10, 2017: Animal Biotechnology
https://www.readbyqxmd.com/read/28631792/nanoscopic-dynamics-of-bicontinous-microemulsions-effect-of-membrane-associated-protein
#6
V K Sharma, Douglas G Hayes, Volker S Urban, Hugh M O'Neill, M Tyagi, E Mamontov
Bicontinous microemulsions (BμE) generally consist of nanodomains formed by surfactant in a mixture of water and oil at nearly equal proportions and are potential candidates for the solubilization and purification of membrane proteins. Here we present the first time report of nanoscopic dynamics of surfactant monolayers within BμEs formed by the anionic surfactant sodium dodecyl sulfate (SDS) measured on the nanosecond to picosecond time scale using quasielastic neutron scattering (QENS). BμEs investigated herein consisted of middle phases isolated from Winsor-III microemulsion systems that were formed by mixing aqueous and oil solutions under optimal conditions...
June 20, 2017: Soft Matter
https://www.readbyqxmd.com/read/28631300/a-bacterial-chloroform-reductive-dehalogenase-purification-and-biochemical-characterization
#7
Bat-Erdene Jugder, Susanne Bohl, Helene Lebhar, Robert D Healey, Mike Manefield, Christopher P Marquis, Matthew Lee
We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF...
June 20, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28630463/the-role-of-antibody-v%C3%AE%C2%BA-framework-3-region-towards-antigen-binding-effects-on-recombinant-production-and-protein-l-binding
#8
Chinh Tran-To Su, Wei-Li Ling, Wai-Heng Lua, Jun-Jie Poh, Samuel Ken-En Gan
Antibody research has traditionally focused on heavy chains, often neglecting the important complementary role of light chains in antibody formation and secretion. In the light chain, the complementarity-determining region 3 (VL-CDR3) is specifically implicated in disease states. By modulating VL-CDR3 exposure on the scaffold through deletions in the framework region 3 (VL-FWR3), we further investigated the effects on secretion in recombinant production and antigen binding kinetics. Our random deletions of two residues in the VL-FWR3 of a Trastuzumab model showed that the single deletions could impact recombinant production without significant effect on Her2 binding...
June 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28630382/immunodiagnostic-value-of-echinococcus-granulosus-recombinant-b8-1-subunit-of-antigen-b
#9
Amir Savardashtaki, Bahador Sarkari, Farzane Arianfar, Zohreh Mostafavi-Pour
BACKGROUND: Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. OBJECTIVE: The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. METHODS: The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized...
June 2017: Iranian Journal of Immunology: IJI
https://www.readbyqxmd.com/read/28628836/an-affinity-based-aqueous-two-phase-mixed-micellar-system-and-its-purification-of-yeast-3-5-bisphosphate-nucleotidase
#10
Yao Liu, Mei-Hao Sun, Shi-Kuan Shao, Gang Deng
Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system...
May 30, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28628608/tcp4-dependent-induction-of-constans-transcription-requires-gigantea-in-photoperiodic-flowering-in-arabidopsis
#11
Akane Kubota, Shogo Ito, Jae Sung Shim, Richard S Johnson, Yong Hun Song, Ghislain Breton, Greg S Goralogia, Michael S Kwon, Dianne Laboy Cintrón, Tomotsugu Koyama, Masaru Ohme-Takagi, Jose L Pruneda-Paz, Steve A Kay, Michael J MacCoss, Takato Imaizumi
Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter...
June 19, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28628207/engineered-polymerases-with-altered-substrate-specificity-expression-and-purification
#12
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28628203/photoactivated-in-vivo-proximity-labeling
#13
David B Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28627725/proteomic-analysis-of-host-cell-protein-dynamics-in-the-supernatant-of-fc-fusion-protein-producing-cho-dg44-and-dukx-b11-cell-lines-in-batch-and-fed-batch-cultures
#14
Jin Hyoung Park, Jong Hwa Jin, In Jung Ji, Hyun Joo An, Jong Won Kim, Gyun Min Lee
Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the commercial production of therapeutic proteins including Fc-fusion proteins. During the culture of recombinant CHO (rCHO) cells, host cell proteins (HCPs), secreted from viable cells and released from dead cells, accumulate extracellularly, potentially impairing product quality. In this study, the HCPs that accumulated extracellularly in batch and fed-batch cultures of Fc-fusion protein-producing rCHO cell lines (DG-Fc and DUKX-Fc) were identified and quantified using nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by gene ontology and functional analysis...
June 19, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28627567/dissecting-binding-of-a-%C3%AE-barrel-membrane-protein-by-phage-display
#15
Luz M Meneghini, Sarvind Tripathi, Marcus A Woodworth, Sudipta Majumdar, Thomas L Poulos, Gregory A Weiss
Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal...
June 19, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28627360/the-plasmodium-falciparum-exported-protein-pf3d7_0402000-binds-to-erythrocyte-ankyrin-and-band-4-1
#16
Bikash Shakya, Wesley D Penn, Ernesto S Nakayasu, Douglas J LaCount
Plasmodium falciparum extensively modifies the infected red blood cell (RBC), resulting in changes in deformability, shape and surface properties. These alterations suggest that the RBC cytoskeleton is a major target for modification during infection. However, the molecular mechanisms leading to these changes are largely unknown. To begin to address this question, we screened for exported P. falciparum proteins that bound to the erythrocyte cytoskeleton proteins ankyrin 1 (ANK1) and band 4.1 (4.1R), which form critical interactions with other cytoskeletal proteins that contribute to the deformability and stability of RBCs...
June 13, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28625912/isolation-of-recombinant-human-untagged-glyceraldehyde-3-phosphate-dehydrogenase-from-e-coli-producer-strain
#17
K V Barinova, M A Eldarov, E V Khomyakova, V I Muronetz, E V Schmalhausen
The goal of the present work was expression of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) without additional tag constructions in E. coli cells and elaboration of the procedure for purification of untagged hGAPDH from the extract of the producer cells. We present a simple method for purification of untagged hGAPDH including ammonium sulfate fractionation and gel filtration on a G-100 Sephadex column. The method allows isolation of 2 mg of pure hGAPDH from 600 ml of cell culture (7 g of the cell biomass)...
June 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28625911/white-shrimp-litopenaeus-vannamei-recombinant-lactate-dehydrogenase-biochemical-and-kinetic-characterization
#18
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/28625565/the-cep19-rabl2-gtpase-complex-binds-ift-b-to-initiate-intraflagellar-transport-at-the-ciliary-base
#19
Tomoharu Kanie, Keene Louis Abbott, Nancie Ann Mooney, Edward Douglas Plowey, Janos Demeter, Peter Kent Jackson
Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes...
June 14, 2017: Developmental Cell
https://www.readbyqxmd.com/read/28624997/expression-and-purification-of-cytochrome-p450-55b1-from-chlamydomonas-reinhardtii-and-its-application-in-nitric-oxide-biosensing
#20
Bin Gong, Xiaosheng Liang, Yong Li, Qian Xiao, Panchun Yang, Yunhua Wu
Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). Here, we expressed the cytochrome P450 55B1 gene with an HIS-tag in E scherichia coli using a pET28a vector. The native protein was produced at a level of 1.59 μmol/g of total protein, with approximately 85% of the P450 being soluble. The CYP55B1 protein was characterized spectrally and purified by a HIS-trap column. This procedure allowed recovery of 45% of the expressed protein and CYP55B1 with a specific content of 0...
June 17, 2017: Applied Biochemistry and Biotechnology
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