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Current Protocols in Protein Science

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https://www.readbyqxmd.com/read/29091276/simple-and-efficient-purification-of-recombinant-proteins-using-the-heparin-binding-affinity-tag
#1
Srinivas Jayanthi, Ravi Kumar Gundampati, Thallapuranam Krishnaswamy Suresh Kumar
Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29091275/visualization-of-protein-interactions-in-living-cells-using-bimolecular-luminescence-complementation-bilc
#2
Lisette G G C Verhoef, Mark Wade
The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed 'bimolecular complementation' relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29091274/screening-fusion-tags-for-improved-recombinant-protein-expression-in-e-coli-with-the-expresso%C3%A2-solubility-and-expression-screening-system
#3
Eric J Steinmetz, Michele E Auldridge
The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29091273/analysis-of-disulfide-bond-formation
#4
Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N Hebert
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29091272/expression-and-purification-of-protein-complexes-suitable-for-structural-studies-using-mammalian-hek-293f-cells
#5
Irene Nigi, Louise Fairall, John W R Schwabe
Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/29091271/screening-and-identifying-membrane-proteins-favorable-for-crystallization
#6
Jared Kim, Ho Leung Ng
This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n-dodecyl-β-maltoside (DDM)...
November 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762495/preparation-of-cell-cultures-and-vaccinia-virus-stocks
#7
Catherine A Cotter, Patricia L Earl, Linda S Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762494/protein-lipid-interaction-by-fluorescence-plif-to-characterize-and-screen-for-inhibitors-of-protein-phosphoinositide-interactions
#8
Laurie Ceccato, Mélanie Mansat, Bernard Payrastre, Frédérique Gaits-Iacovoni, Julien Viaud
Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities...
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762493/mass-tag-labeling-using-acyl-peg-exchange-for-the-determination-of-endogenous-protein-s-fatty-acylation
#9
Avital Percher, Emmanuelle Thinon, Howard Hang
The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S-fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S-fatty acylation, these are hampered in their ability to characterize endogenous protein S-fatty acylation levels under physiological conditions...
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762492/purification-of-recombinant-human-tyrosinase-from-insect-larvae-infected-with-the-baculovirus-vector
#10
Monika B Dolinska, Paul T Wingfield, Yuri V Sergeev
The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography...
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762491/generation-of-recombinant-vaccinia-viruses
#11
Linda S Wyatt, Patricia L Earl, Bernard Moss
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28762490/site-specific-protein-labeling-via-sortase-mediated-transpeptidation
#12
John M Antos, Jessica Ingram, Tao Fang, Novalia Pishesha, Matthias C Truttmann, Hidde L Ploegh
Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles...
August 1, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369669/bioluminescence-resonance-energy-transfer-bret-based-synthetic-sensor-platform-for-drug-discovery
#13
Jongchan Woo, Jason Hong, Savithramma P Dinesh-Kumar
Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein-protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro...
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369668/selective-proteomic-proximity-labeling-assay-using-tyramide-spplat-a-quantitative-method-for-the-proteomic-analysis-of-localized-membrane-bound-protein-clusters
#14
Johanna Susan Rees, Xue-Wen Li, Sarah Perrett, Kathryn Susan Lilley, Antony Philip Jackson
This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target...
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369667/measuring-protein-interactions-by-optical-biosensors
#15
Huaying Zhao, Lisa F Boyd, Peter Schuck
This unit gives an introduction to the basic techniques of optical biosensing for measuring equilibrium and kinetics of reversible protein interactions. Emphasis is placed on description of robust approaches that will provide reliable results with few assumptions. How to avoid the most commonly encountered problems and artifacts is also discussed. © 2017 by John Wiley & Sons, Inc.
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369666/computational-prediction-of-intrinsic-disorder-in-proteins
#16
Fanchi Meng, Vladimir Uversky, Lukasz Kurgan
Computational prediction of intrinsically disordered proteins (IDPs) is a mature research field. These methods predict disordered residues and regions in an input protein chain. More than 60 predictors of IDPs have been developed. This unit defines computational prediction of intrinsic disorder, summarizes major types of predictors of disorder, and provides details about three accurate and recently released methods. We demonstrate the use of these methods to predict intrinsic disorder for several illustrative proteins, provide insights into how predictions should be interpreted, and quantify and discuss predictive performance...
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369665/immunoblotting-and-immunodetection
#17
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28369664/n-terminal-methionine-processing
#18
Paul T Wingfield
Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1...
April 3, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28150884/characterization-of-protein-content-present-in-exosomes-isolated-from-conditioned-media-and-urine
#19
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 packaged as cargo molecules within vesicles that are released to the extracellular space (termed extracellular vesicles or EVs). A subclass of such EVs, exosomes have been shown to horizontally transfer information...
February 2, 2017: Current Protocols in Protein Science
https://www.readbyqxmd.com/read/28150883/analysis-of-protein-o-glcnacylation-by-mass-spectrometry
#20
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 chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O-GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O-GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision-induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]...
February 2, 2017: Current Protocols in Protein Science
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