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Antibody purification

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https://www.readbyqxmd.com/read/28439867/mapping-protein-protein-interactions-using-affinity-purification-and-mass-spectrometry
#1
Chin-Mei Lee, Christopher Adamchek, Ann Feke, Dmitri A Nusinow, Joshua M Gendron
The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28431943/expression-and-purification-of-p70%C3%AE-ct104-s6-k-a-72-kda-c-terminal-truncated-p70s6-kinase-gst-fusion-protein-in-bacterial-expression-system
#2
Younis Mohammad Hazari, Irfana Reshi, Mudasir Habib, Khalid Majid Fazili
The p70ΔCT104 S6K is a 421 amino acid residue long truncated form of p70S6 kinase, with 104 amino acids residues cleaved from the carboxyl terminal end of the original protein. The p70ΔCT104 S6K was cloned in E. coli DH5α and successfully expressed in E. coli BL21 (DE3) strain. Western blot with rabbit polyclonal anti-GST antibody was used to follow the protein during expression and purification. The protein purification was achieved by affinity chromatography using Glutathione resin-agarose beads, followed by chromatography on a spin concentration column...
April 18, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28429162/involvement-of-methylated-hbha-expressed-from-mycobacterium-smegmatis-in-an-ifn-%C3%AE-release-assay-to-aid-discrimination-between-latent-infection-and-active-tuberculosis-in-bcg-vaccinated-populations
#3
H-L Wen, C-L Li, G Li, Y-H Lu, H-C Li, T Li, H-M Zhao, K Wu, D B Lowrie, J-X Lv, S-H Lu, X-Y Fan
IFN-γ release assays (IGRAs) based on region of difference 1 (RD1) antigens have improved diagnosis of Mycobacterium tuberculosis (M. tb) infection. However, IGRAs with these antigens cannot discriminate between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). M. tb heparin-binding-hemagglutinin (HBHA) induces relatively high IFN-γ responses in LTBI individuals and low responses in ATB patients, but purification of the native methylated HBHA from cultures of M. tb for immunological tests is complex and time-consuming...
April 20, 2017: European Journal of Clinical Microbiology & Infectious Diseases
https://www.readbyqxmd.com/read/28428153/development-of-polyol-responsive-antibody-mimetics-for-single-step-protein-purification
#4
Richard J Suderman, Daren A Rice, Shane D Gibson, Eric J Strick, David M Chao
The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity...
April 17, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28425268/efficient-production-of-nanobodies-against-urease-activity-ofhelicobacter-pylori-in-pichia-pastoris
#5
Shahrbanoo Pourasadi, Seyed Latif Mousavi Gargari, Masoumeh Rajabibazl, Shahram Nazarian
BACKGROUND/AIM: Helicobacter pylori is a major health problem. One of the therapeutic approaches is administration of antibody against H. pylori. The methylotrophic Pichia pastoris is a suitable host for expression of recombinant antibody fragments. The aims of this study were the expression and the evaluation of camelid nanobody in the yeast Pichia pastoris. MATERIALS AND METHODS: The camelid-derived heavy-chain antibody (nanobody) against the UreC subunit of urease from H...
April 18, 2017: Turkish Journal of Medical Sciences
https://www.readbyqxmd.com/read/28421663/cloning-and-plant-based-production-of-antibody-mc10e7-for-a-lateral-flow-immunoassay-to-detect-4-arginine-microcystin-in-fresh-water
#6
Stanislav Melnik, A-C Neumann, R Karongo, S Dirndorfer, Martin Stübler, Verena Ibl, R Niessner, Dietmar Knopp, Eva Stoger
Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full size version in leaves of Nicotiana tabacum and N...
April 19, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28421414/polyclonal-antibodies-in-microplates-to-predict-the-maximum-adsorption-activities-of-enzyme-mutants-from-cell-lysates
#7
Yiran Feng, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, Huimin Chong, Juan Liao, Chang-Guo Zhan, Fei Liao
With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs...
April 19, 2017: Protein Journal
https://www.readbyqxmd.com/read/28419935/identification-of-a-host-cell-protein-impurity-in-therapeutic-protein-p1
#8
Deepti Ahluwalia, Harbhajan Dhillon, Thomas Slaney, Hangtian Song, Heather Boux, Sheetal Mehta, Lei Zhang, Anulfo Valdeza, Girija Krishnamurthy
Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification...
April 9, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28417626/production-and-characterization-of-monoclonal-antibody-against-recombinant-virus-coat-protein-cp42
#9
Naeimeh Shibaei, Jafar Majidi, Khadijeh Razavi, Ali Asghar Karkhane, Nemat Sokhandan-Bashir, Leili Aghebati-Maleki
There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E...
February 2017: Iranian Journal of Allergy, Asthma, and Immunology
https://www.readbyqxmd.com/read/28415641/identification-and-screening-of-effective-protective-antigens-for-channel-catfish-against-streptococcus-iniae
#10
Yajun Wang, Erlong Wang, Yang He, Kaiyu Wang, Qian Yang, Jun Wang, Yi Geng, Defang Chen, Xiaoli Huang, Ping Ouyang, Weimin Lai, Cunbin Shi
Vaccination is a potential approach for prevention and control of disease in fish. The use of genetically engineered vaccines is an effective method and a green intervention to control bacterial infection in aquaculture. However, efforts to develop these vaccines are limited by the lack of conserved protective antigens. In this study, three candidate immunogens (Srr, NeuA, and Hsp) of the pathogenic Streptococcus iniae strain DGX07 isolated from diseased channel catfish were identified and analyzed. Molecular cloning, expression, and purification of candidate antigen genes were carried out to obtain the candidate immunogens in the form of recombinant subunit vaccines...
March 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/28414907/cell-isolation-and-recovery-using-hollow-glass-microspheres-coated-with-nanolayered-films-for-applications-in-resource-limited-settings
#11
Ziye Dong, Caroline C Ahrens, Dan Yu, Zhenya Ding, HyunTaek Lim, Wei Li
Established cell isolation and purification techniques such as FACS, isolation through magnetic micro/nano-particles and recovery via microfluidic devices have limited application as disposable technologies appropriate for point-of-care use in remote areas where lab equipment as well as electrical, magnetic, and optical sources are restricted. We report a simple yet effective method for cell isolation and recovery that requires neither specialized lab equipment nor any form of power source. Specifically, self-floating hollow glass microspheres were coated with an enzymatically degradable nanolayered film, and conjugated with antibodies to allow both fast capture and release of subpopulations of cells from a cell mixture...
April 17, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28414239/a-novel-sample-preparation-for-shotgun-proteomics-characterization-of-hcps-in-antibodies
#12
Lihua Huang, Ning Wang, Charles E Mitchell, Tammy J Brownlee, Steven R Maple, Michael R De Felippis
Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection and quantitation purposes is most commonly used to monitor HCP removal during process development but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development...
April 17, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28413818/data-on-enhanced-expression-and-purification-of-camelid-single-domain-antibodies-from-escherichia-coli-classical-inclusion-bodies
#13
Maristella Maggi, Claudia Scotti
Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells...
June 2017: Data in Brief
https://www.readbyqxmd.com/read/28412509/a-monoclonal-antibody-recognizes-undifferentiation-specific-carbohydrate-moieties-expressed-on-cell-surface-of-the-human-dental-pulp-cells
#14
Kyung-Jung Kang, Seon-Yle Ko, Chun-Jeih Ryu, Young-Joo Jang
Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization...
April 6, 2017: Stem Cell Research
https://www.readbyqxmd.com/read/28410804/neutron-reflectivity-measurement-of-protein-a-antibody-complex-at-the-solid-liquid-interface
#15
Alice R Mazzer, Luke A Clifton, Tatiana Perevozchikova, Paul D Butler, Christopher J Roberts, Daniel G Bracewell
Chromatography is a ubiquitous unit operation in the purification of biopharmaceuticals yet few studies have addressed the biophysical characterisation of proteins at the solution-resin interface. Chromatography and other adsorption and desorption processes have been shown to induce protein aggregation which is undesirable in biopharmaceutical products. In order to advance understanding of how adsorption processes might impact protein stability, neutron reflectivity was used to characterise the structure of adsorbed immunoglobulin G (IgG) on model surfaces...
April 1, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28409449/isolation-of-native-soluble-and-membrane-bound-protein-complexes-from-yeast-saccharomyces-cerevisiae
#16
Tobias Hansen, Anna Chan, Thomas Schröter, Daniel Schwerter, Wolfgang Girzalsky, Ralf Erdmann
Immunoprecipitation is a traditional approach to isolate single proteins or native protein complexes from a complex sample mixture. The original method makes use of specific antibodies against endogenous proteins or epitope tags, which are first bound to the target protein and then isolated with protein A beads. An advancement of this method is the application of a protein A tag fused to the target protein and the affinity-purification of the tagged protein with human Immunoglobulin G chemically cross-linked to a sepharose matrix...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28402653/host-cell-protein-profiling-by-targeted-and-untargeted-analysis-of-data-independent-acquisition-mass-spectrometry-data-with-parallel-reaction-monitoring-verification
#17
Simion Kreimer, Yuanwei Gao, Somak Ray, Mi Jin, Zhijun Tan, Nesredin A Mussa, Li Tao, Zhengjian Li, Alexander R Ivanov, Barry L Karger
Host cell proteins (HCPs) are process-related impurities of biopharmaceuticals that remain at trace levels despite multiple stages of downstream purification. Currently, there is interest in implementing LC-MS in biopharmaceutical HCP profiling alongside conventional ELISA, because individual species can be identified and quantitated. Conventional data-dependent LC-MS is hampered by the low concentration of HCP-derived peptides, which are 5-6 orders of magnitude less abundant than the biopharmaceutical-derived peptides...
April 12, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28402142/development-of-separation-technology-for-the-removal-of-radium-223-from-targeted-thorium-conjugate-formulations-part-ii-purification-of-targeted-thorium-conjugates-on-cation-exchange-columns
#18
Janne Olsen Frenvik, Knut Dyrstad, Solveig Kristensen, Olav B Ryan
Tumour targeting pharmaceuticals will play a crucial role in future pharma pipelines. The Targeted Thorium Conjugate (TTC) therapeutic platform could provide real benefit to patients, whereby targeting moieties like monoclonal antibodies are radiolabelled with the alpha-emitting radionuclide thorium-227 ((227)Th, t1/2=18.7 days). A potential problem could be the accumulation of the long-lived daughter nuclide radium-223 ((223)Ra, t1/2=11.4 days) in the drug product during manufacture and distribution. Therefore, the level of (223)Ra must be standardised before administration to the patient...
April 12, 2017: Drug Development and Industrial Pharmacy
https://www.readbyqxmd.com/read/28394698/generation-of-therapeutic-immunoconjugates-via-residue-specific-conjugation-technology-respect-utilizing-a-native-cysteine-in-the-light-chain-framework-of-oryctolagus-cuniculus
#19
Earl F Albone, Jared L Spidel, Xin Cheng, Young Chul Park, Sara Jacob, Andrew Z Milinichik, Ben Vassen, Janet Butler, J Bradford Kline, Luigi Grasso
The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be utilized for site-specific conjugation...
April 10, 2017: Cancer Biology & Therapy
https://www.readbyqxmd.com/read/28389350/process-development-for-production-and-purification-of-the-schistosoma-mansoni-sm14-antigen
#20
Leonardo Damasceno, Gerd Ritter, Carl A Batt
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L(-1). Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter...
April 5, 2017: Protein Expression and Purification
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