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Antibody purification

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https://www.readbyqxmd.com/read/28339303/development-of-rap-tag-a-novel-tagging-system-for-protein-detection-and-purification
#1
Yuki Fujii, Mika K Kaneko, Satoshi Ogasawara, Shinji Yamada, Miyuki Yanaka, Takuro Nakamura, Noriko Saidoh, Kanae Yoshida, Ryusuke Honma, Yukinari Kato
Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification...
March 24, 2017: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
https://www.readbyqxmd.com/read/28330270/expression-and-purification-of-a-gene-encoding-a-9-7%C3%A2-kda-pe-protein-of-mycobacterium-avium-subsp-paratuberculosis
#2
S Chandra Sekar, P P Goswami, R Deb
Mycobacterium avium subsp. paratuberculosis (Map) contains PE family antigens which are Proline and glutamic acid rich and may play important role as T-cell antigens. In the present study, the Map 1507 ORF encoding 9.7 kDa PE protein was amplified by polymerase chain reaction and cloned into E. coli vector pQE30 UA. The recombinant plasmid designated as pQ PE was transformed into E. coli M15 cells and induced with IPTG revealed the high level expression of 11.9 kDa His-fusion protein as estimated by migration in 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...
December 2016: 3 Biotech
https://www.readbyqxmd.com/read/28325282/dna-aptamers-for-the-characterization-of-histological-structure-of-lung-adenocarcinoma
#3
Galina S Zamay, Tatiana I Ivanchenko, Tatiana N Zamay, Valentina L Grigorieva, Yury E Glazyrin, Olga S Kolovskaya, Irina V Garanzha, Andrey A Barinov, Alexey V Krat, Gleb G Mironov, Ana Gargaun, Dmitry V Veprintsev, Sergey S Bekuzarov, Andrey K Kirichenko, Ruslan A Zukov, Marina M Petrova, Andrey A Modestov, Maxim V Berezovski, Anna S Zamay
Nucleic acid aptamers are becoming popular as molecular probes for identification and imaging pathology and, at the same time, as a convenient platform for targeted therapy. Recent studies have shown that aptamers may be effectively used for tumor characterization and as commercially available monoclonal antibodies. Here we present three DNA aptamers binding to whole transformed lung cancer tissues, including tumor cells, connective tissues, and blood vessels. Protein targets have been revealed using affinity purification followed by mass spectrometry analyses, and they have been validated using a panel of correspondent antibodies and 3D imaging of tumor tissues...
March 17, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28323416/one-pot-two-nanoprobe-assay-uncovers-targeted-glycoprotein-biosignature
#4
Mira Anne C Dela Rosa, Wei-Chun Chen, Yi-Ju Chen, Rofeamor P Obena, Chih-Hsiang Chang, Rey Y Capangpangan, Tung-Hung Su, Chi-Ling Chen, Pei-Jer Chen, Yu-Ju Chen
We report a one-pot two-nanoprobe approach coupled to mass spectrometry for simultaneous quantification and post-translational modification (PTM) profiling of targeted protein in biofluid. Using N-glycoprotein as model, the assay employs two nanoprobes, antibody-conjugated SiO2 nanoparticles and lectin-conjugated magnetic Fe3O4 nanoparticles, to achieve target glycoprotein isolation from biofluid and subsequent glycopeptide enrichment in a single tube. As demonstrated on α-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma (HCC), the assay has high purification specificity (20 glycopeptides) with 2-fold and 10-fold superior total glycopeptide intensity compared to non-one-pot method (9 glycopeptides) or without enrichment (6 glycopeptides), respectively...
March 21, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28322788/a-recombinant-polypeptide-of-the-megakaryocyte-potentiating-factor-is-a-potential-biomarker-in-plasma-for-the-detection-of-mesothelioma
#5
Irina Raiko, Hans-Peter Rihs, Jan Gleichenhagen, Ingrid Sander, Jens Kollmeier, Martin Lehnert, Thomas Brüning, Georg Johnen
Malignant mesothelioma (MM) is a fatal disease mostly associated with asbestos exposure and difficult to detect by non-invasive methods. This study aimed to use recombinant fragments of the megakaryocyte potentiating factor (MPF) for the development of cost-effective MPF ELISAs. Three polypeptides spanning the MPF region (MPF1-148, MPF 34-288, MPF/MSLN254-400) were produced in E.coli as maltose-binding protein hybrids. After isolation, Factor Xa digest, and purification, the polypeptides were used for the generation of rabbit antibodies and development of ELISAs...
March 17, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28318569/designing-affinity-chromatographic-processes-for-the-capture-of-antibodies-part-i-a-simplified-approach
#6
D Pfister, L David, M Holzer, R-M Nicoud
Protein A affinity chromatography is a standard technique for the purification of therapeutic antibodies. Recently, multi-column chromatographic processes have emerged to turn classical capture processes into more efficient and continuous systems. The design of such chromatographic processes, be they single-column or multi-column systems, is described in this work. A rational method to conceive, scale-up and compare processes is proposed and illustrated with different examples. All along this article, the results of the equilibrium theory, i...
March 2, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28317180/anti-acsa-2-defines-a-novel-monoclonal-antibody-for-prospective-isolation-of-living-neonatal-and-adult-astrocytes
#7
Christina G Kantzer, Camille Boutin, Ina D Herzig, Carolina Wittwer, Sandy Reiß, Marie Catherine Tiveron, Jan Drewes, Thomas D Rockel, Stefanie Ohlig, Jovica Ninkovic, Harold Cremer, Sandra Pennartz, Melanie Jungblut, Andreas Bosio
Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2(+) cells, microglia, endothelial cells, leukocytes, or erythrocytes...
March 20, 2017: Glia
https://www.readbyqxmd.com/read/28316235/evaluation-of-nanobody-conjugates-and-protein-fusions-as-bioanalytical-reagents
#8
Virginia J Bruce, Brian R McNaughton
Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution traditionally requires the use of one or more commercially available antibody-small-molecule dye, or antibody-reporter protein conjugates that recognize relatively short peptide tags (<15 amino acids). However, the size of antibodies, and their molecular complexity (by virtue of post-translational disulfide formation and glycosylation) typically requires either expression in mammalian cells or purification from immunized mammals...
March 20, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28315746/optimized-protocol-for-soluble-prokaryotic-expression-purification-and-structural-analysis-of-human-placenta-specific-1-plac1
#9
Mahboobeh Nazari, Amir-Hassan Zarnani, Roya Ghods, Rahman Emamzadeh, Somayeh Najafzadeh, Arash Minai-Tehrani, Jafar Mahmoudian, Maryam Yousefi, Sedigheh Vafaei, Sam Massahi, Mohammad-Reza Nejadmoghaddam
Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles...
March 16, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28314374/hiv-1-consensus-envelope-induced-broadly-binding-antibodies
#10
R Ryan Meyerhoff, Richard M Scearce, Damon F Ogburn, Brad Lockwood, Joy Pickeral, Masa Kuraoka, Kara Anasti, Joshua Eudailey, Amanda Eaton, Melissa Cooper, Kevin Wiehe, David C Montefiori, Georgia D Tomaras, Guido Ferrari, S Munir Alam, Hua-Xin Liao, Bette Korber, Feng Gao, Barton F Haynes
Antibodies that cross-react with multiple HIV-1 envelopes (Envs) are useful reagents for characterizing Env proteins and for soluble Env capture and purification assays. We previously reported ten murine monoclonal antibodies induced by group M consensus Env, CON-6 immunization. Each demonstrated broad cross-reactivity to recombinant Envs. Here we characterized the Env epitopes to which they bind. Seven mapped to linear epitopes in gp120, five at the Env N-terminus and two at the Env C-terminus. One antibody, 13D7, bound at the gp120 N-terminus (aa 30-42), reacted with HIV-1-infected CD4+ T cells, and when expressed in a human IgG1 backbone, mediated ADCC...
March 17, 2017: AIDS Research and Human Retroviruses
https://www.readbyqxmd.com/read/28303322/super-sensitive-time-resolved-fluoroimmunoassay-for-thyroid-stimulating-hormone-utilizing-europium-iii-nanoparticle-labels-achieved-by-protein-corona-stabilization-short-binding-time-and-serum-preprocessing
#11
Tuomas Näreoja, Jessica M Rosenholm, Urpo Lamminmäki, Pekka E Hänninen
Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L(-1) in preprocessed serum samples by reducing nonspecific binding...
March 16, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28293231/isolation-of-francisella-tularensis-and-yersinia-pestis-from-blood-cultures-by-plasma-purification-and-immunomagnetic-separation-accelerates-antibiotic-susceptibility-determination
#12
Ronit Aloni-Grinstein, Ofir Schuster, Shmuel Yitzhaki, Moshe Aftalion, Sharon Maoz, Ida Steinberger-Levy, Raphael Ber
The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28283246/experimental-design-of-a-twin-column-countercurrent-gradient-purification-process
#13
Fabian Steinebach, Nicole Ulmer, Lara Decker, Lars Aumann, Massimo Morbidelli
As typical for separation processes, single unit batch chromatography exhibits a trade-off between purity and yield. The twin-column MCSGP (multi-column countercurrent solvent gradient purification) process allows alleviating such trade-offs, particularly in the case of difficult separations. In this work an efficient and reliable procedure for the design of the twin-column MCSGP process is developed. This is based on a single batch chromatogram, which is selected as the design chromatogram. The derived MCSGP operation is not intended to provide optimal performance, but it provides the target product in the selected fraction of the batch chromatogram, but with higher yield...
February 23, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28283009/-production-and-molecular-characterization-of-plasmodium-falciparum-recombinant-circumsporozoite-protein-with-37-nanp-and-4-nvdp-epitopes
#14
Yunus Uyar, Abdüssamed Akşit, Serkan Karaca, Şirin Sahra Ceylan, Merve Yürük
Malaria is caused by the protozoan parasite Plasmodium, the leading cause of death amongst the parasitic diseases. The disease is transmitted to human by the bites of female Anopheles mosquitoes. According to the World Health Organization (WHO) data, there were an estimated 214 million malaria cases and estimated 438.000 deaths occurred worldwide, in 2015. It is observed that 90% of all the deaths due to malaria occur in Africa. 78% of these cases were children who are under five years old. Intensive malaria interventions helped to reduce malaria incidence by 37% between 2000 and 2015...
January 2017: Mikrobiyoloji Bülteni
https://www.readbyqxmd.com/read/28281648/proteomic-analysis-of-host-cell-protein-dynamics-in-the-culture-supernatants-of-antibody-producing-cho-cells
#15
Jin Hyoung Park, Jong Hwa Jin, Myung Sin Lim, Hyun Joo An, Jong Won Kim, Gyun Min Lee
Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis...
March 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28279788/containment-challenges-in-hpapi-manufacture-for-adc-generation
#16
REVIEW
Elizabeth Dunny, Imelda O'Connor, Jonathan Bones
Antibody-drug conjugates (ADCs) are emerging as an impactful class of therapeutics for the treatment of cancer because of their ability to harness the specificity of an antibody and the cytotoxic potential of the payload to target and destroy cancer cells. However, the potent nature of the cytotoxic payload creates associated manufacturing challenges for active pharmaceutical ingredient (API) manufacturers, because huge investment in containment technology is required to ensure the protection of operators and the environment...
March 6, 2017: Drug Discovery Today
https://www.readbyqxmd.com/read/28275789/-preparation-and-identification-of-polyclonal-antibody-of-streptococcus-salivarius-57-i-urease
#17
Yan Wang, Dan-Ying Tao, Xi-Ping Feng
PURPOSE: To prepare and identify polyclonal antibody of Streptococcus salivarius 57.I urease. METHODS: The biggest structural subunit of Streptococcus salivarius 57.I urease, UreC, was obtained by gene clone, IPTG-induced expression, and purification through affinity chromatography. Anti-sera and polyclonal antibody were raised by immunizing rabbits with purified UreC. Western blot was utilized to detect the specific combination of polyclonal antibody with UreC...
December 2016: Shanghai Kou Qiang Yi Xue, Shanghai Journal of Stomatology
https://www.readbyqxmd.com/read/28269763/development-of-high-affinity-monoclonal-antibody-using-cd44-overexpressed-cells-as-a-candidate-for-targeted-immunotherapy-and-diagnosis-of-acute-myeloid-leukemia
#18
Amir Amanzadeh, Fatemeh Heidarnejad, Meghdad Abdollahpour-Alitappeh, Vahid Molla Kazemiha, Shamsi Yari, Ali Reza Hadizadeh Tasbiti, Mahdi Habibi Anbouhi, Mohsen Abolhassani, Mohammad Ali Shokrgozar
AIM: CD44s antigens have been suggested as an efficient biomarker for cancer stem cells. Current study aimed to develop a hybridoma that producing a high affinity murine anti-human CD44 monoclonal antibody for early diagnostic laboratory tests of some cancer. MATERIALS AND METHODS: To make hybridoma against CD44, mice were immunized with MDA-MB-468 cells. Resulted hybridomas using three culture media were screened by indirect ELISA, then cloned by limiting dilution, and isotype was determined after obtaining ascitic fluid and antibody purification...
February 25, 2017: Human Antibodies
https://www.readbyqxmd.com/read/28260372/large-scale-filter-aided-sample-preparation-method-for-the-analysis-of-the-ubiquitinome
#19
Albert Casanovas, Roberto Pinto-Llorente, Montserrat Carrascal, Joaquin Abian
Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment...
March 16, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28255878/utilization-of-selenocysteine-for-site-specific-antibody-conjugation
#20
Xiuling Li, Christoph Rader
Site-specific conjugation methods are becoming increasingly important in building next-generation antibody-drug conjugates. We have developed a site-specific conjugation technology based on monoclonal antibodies with engineered selenocysteine (Sec) residues, named selenomabs. Here, we provide protocols for the engineering, expression, and purification of selenomabs in single-chain variable fragment (scFv)-Fc format. Methods for selective conjugation of selenomabs to selenol-reactive compounds and analytical characterization of selenomab conjugates are also included...
2017: Methods in Molecular Biology
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