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Isotype Determination of Antibodies
Peter Hornbeck, Thomas A. Fleisher, Nicholas M. Papadopoulos
On identifying a new monoclonal antibody, or in characterizing antibodies in sera evoked by disease or immunization, it is particularly informative to determine the serological class of the antibodies. The serological class of the protein is determined by the structure of the antibody constant
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Double‐Immunodiffusion Assay for Detecting Specific Antibodies (Ouchterlony)
Peter Hornbeck
The method first described by Ouchterlony in 1948 is a classic and simple technique that permits evaluation and comparison of antibodies in animal or human sera directed against protein or complex carbohydrate antigens. It is a low‐tech procedure that may provide information on the relative quantity
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Web Sites of Interest to Immunologists
Marie‐Paule Lefranc
This appendix includes a selection of links of interest to immunologists from the international ImMunoGeneTics (IMGT) database, covering a wide variety of subject matter. © 2017 by John Wiley & Sons, Inc. Keywords: ImMunoGeneTics; IMGT; database; immunology; immunoinformatics
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Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Sean R. Gallagher
Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing
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Virus Hunting: Discovery of New Episomal Circular Viruses by Rolling Circle Techniques
Bert Vanmechelen, Annabel Rector, Piet Maes
Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the
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Actinomadura Species: Laboratory Maintenance and Ribosome Engineering
Dipesh Dhakal, Nguyen Thanh Chung, Vijay Rayamajhi, Jae Kyung Sohng
Actinomadura spp. are aerobic, Gram‐positive, catalase‐positive, non‐acid fast, non‐motile actinomycetes. Some species of Actinomadura are associated with opportunistic infections in humans. However, many bioactive compounds with pharmaceutical applications can be isolated from various Actinomadura
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Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites
Dipesh Dhakal, Anaya Raj Pokhrel, Amit Kumar Jha, Nguyen Huy Thuan, Jae Kyung Sohng
Saccharopolyspora spp. are aerobic, Gram‐positive, non‐acid‐fast, and non‐motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for
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Cryptosporidium: Identification and Genetic Typing
Jan Šlapeta
Cryptosporidium spp. are obligate protozoan parasites of the gastrointestinal tract of vertebrates, including humans. In the majority of human cases, the diarrheal disease cryptosporidiosis is caused by either the human‐adapted species Cryptosporidium hominis or the zoonotic Cryptosporidium parvum
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Toxoplasma gondii: Laboratory Maintenance and Growth
Asis Khan, Michael E. Grigg
Toxoplasma gondii is a highly successful apicomplexan protozoan capable of infecting any warm‐blooded animal worldwide. In humans, Toxoplasma infections are life‐long, with approximately one‐third of the world's population chronically infected. Although normally controlled by the host immune system,
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Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9
Yufeng Fang, Linkai Cui, Biao Gu, Felipe Arredondo, Brett M. Tyler
Phytophthora is a filamentous fungus‐like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion‐dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has
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Assays for Acetylation and Other Acylations of Lysine Residues
Nadine Pelletier, Serge Grégoire, Xiang‐Jiao Yang
Lysine acetylation refers to addition of an acetyl moiety to the epsilon‐amino group of a lysine residue and is important for regulating protein functions in various organisms from bacteria to humans. This is a reversible and precisely controlled covalent modification that either serves as an on/off
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Detection and Measurement of Methionine Oxidation in Proteins
K. Ilker Sen, Robert Hepler, Hirsh Nanda
Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In
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Modification of Cysteine
Gregory A. Grant
This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N ‐ethylmaleimide (NEM) is presented, and alternate protocols describe
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Non‐Gradient Blue Native Polyacrylamide Gel Electrophoresis
Xiaoting Luo, Jinzi Wu, Zhen Jin, Liang‐Jun Yan
Gradient blue native polyacrylamide gel electrophoresis (BN‐PAGE) is a well established and widely used technique for activity analysis of high‐molecular‐weight proteins, protein complexes, and protein‐protein interactions. Since its inception in the early 1990s, a variety of minor modifications
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Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine
Ankit Sinha, Javier Alfaro, Thomas Kislinger
Cells secrete biomolecules into the extracellular space as a way of intercellular communication. Secreted proteins can act as ligands that engage specific receptors—on the same cell, nearby cells, or distant cells—and induce defined signaling pathways. Proteins and other biomolecules can also be
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Analysis of Protein O‐GlcNAcylation by Mass Spectrometry
Junfeng Ma, Gerald W. Hart
O‐linked β‐ D ‐ N ‐acetyl glucosamine (O‐GlcNAc) addition (O‐GlcNAcylation), a post‐translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O‐GlcNAcylation underlies the initiation and progression of multiple
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Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting
Ulf Diekmann, Claudia Davenport, Jasmin Kresse, Ortwin Naujok
Pluripotent stem cells have the capability to differentiate into any somatic cell type of the human body. The generation of surrogate cells for the treatment of liver, lung, and pancreatic diseases is of great medical interest. First, the in vitro formation into cells of the definitive endoderm is
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Efficient Generation of Cardiac Purkinje‐like Cells from Embryonic Stem Cells by Activating cAMP Signaling
Su‐Yi Tsai, Shuibing Chen, Todd Evans
Strategies to derive cardiac conduction system (CCS) cells including Purkinje cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. However, using current cardiac differentiation protocols, the
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Magnetic Nanoparticle‐Mediated Gene Delivery to Two‐ and Three‐Dimensional Neural Stem Cell Cultures: Magnet‐Assisted Transfection and Multifection Approaches to Enhance Outcomes
Mark R. Pickard, Christopher F. Adams, Divya M. Chari
Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non‐viral vectors for safe
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Reprogramming of Pancreatic Acinar Cells to Functional Beta Cells by In Vivo Transduction of a Polycistronic Construct Containing Pdx1, Ngn3, MafA in Mice
C. Cavelti‐Weder, A. Zumsteg, W. Li, Q. Zhou
To generate new beta cells after birth is a key focus of regenerative medicine, which could greatly aid the major health burden of diabetes. Beta‐cell regeneration has been described using four different approaches: (1) the development of beta cells from putative precursor cells of the adult
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Flow‐Cytometry‐Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA‐Based Probe
Yong Xue, Jon G. Wilkes, Ted J. Moskal, Anna J. Williams, Willie M. Cooper, Dan A. Buzatu
Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time‐consuming procedures, which include multiple culture incubation steps, and require
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Assessment of Bile Salt Export Pump (BSEP) Inhibition in Membrane Vesicles Using Radioactive and LC/MS‐Based Detection Methods
Lisa D. Marroquin, Paul D. Bonin, Julie Keefer, Thomas Schroeter
The bile salt export pump (BSEP, ABCB11) belongs to the ATP‐binding‐cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in
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Detecting Protein Sulfenylation in Cells Exposed to a Toxicant
Phillip A. Wages
Protein sulfenylation is a post‐translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide
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Detection and Manipulation of the Stress Response Protein Metallothionein
Sadikshya Bhandari, Clare Melchiorre, Kristen Dostie, Debby Laukens, Lindsey Devisscher, Ariel Louwrier, Amy Thees, Michael A. Lynes
Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been
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Preparation of Specific Compartments of the Lungs for Pathologic and Biochemical Analysis of Toxicologic Responses
Laura S. Van Winkle, Jacklyn S. Kelty, Charles G. Plopper
This unit focuses on protocols for assessing microenvironment‐specific responses in the thoracic lung tissues. Aspects of the entire respiratory system serve as potential targets for candidate toxicants, but each candidate toxicant may impact distinct sites due to differential distribution of either
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