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Current Protocols in Protein Science

Sandra Harper, David W Speicher
This manuscript describes protocols for separation of complex protein samples using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Electrophoresis in a single dimension, e.g., 1D SDS polyacrylamide gels, has the potential to rapidly separate hundreds of proteins. When two orthogonal high-resolution electrophoretic methods are efficiently combined in perpendicular dimensions, complex protein mixtures can be separated into thousands of discrete spots. The most common 2D gel separation for intact proteins involves a first-dimensional separation using isoelectric focusing (IEF) followed by separation based on protein size (SDS-PAGE)...
March 6, 2019: Current Protocols in Protein Science
Hsin-Yao Tang, David W Speicher
The formation of disulfide bonds in proteins is an important post-translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified -SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence...
February 12, 2019: Current Protocols in Protein Science
Guillaume Adelmant, Brijesh K Garg, Maria Tavares, Joseph D Card, Jarrod A Marto
Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
February 1, 2019: Current Protocols in Protein Science
Shanmugasundaram Ganapathy-Kanniappan
The electrophoretic mobility of a protein on an immobilized pH-gradient gel (IPG) depends upon its overall positive (acidic) or negative (basic) charge, the principle underlying the IEF technique. In isoelectrofocusing (IEF), a protein with a net positive or negative charge migrates through the pH gradient gel until it reaches the isoelectric point (pI), a pH at which it remains neutral. Thus, the pI of a protein indicates its net charge, a critical determinant of its stability/activity in a given milieu. Conventionally, the first-dimensional IPG-IEF is followed by a second dimension, by which the focused proteins are denatured/reduced and resolved on an SDS-PAGE gel for subsequent immunoblotting to verify the protein identity...
January 31, 2019: Current Protocols in Protein Science
Alexei Yeliseev
Cannabinoid receptor type II, or CB2 , is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB2 is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB2 is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB2 and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR)...
January 9, 2019: Current Protocols in Protein Science
Paschalis-Thomas Doulias, Neal S Gould
The wide reactivity of the thiol group enables the formation of a variety of reversible, covalent modifications on cysteine residues. S-nitrosylation, like many other post-translational modifications, is site selective, reversible, and necessary for a wide variety of fundamental cellular processes. The overall abundance of S-nitrosylated proteins and reactivity of the nitrosyl group necessitates an enrichment strategy for accurate detection with adequate depth. Herein, a method is presented for the enrichment and detection of endogenous protein S-nitrosylation from complex mixtures of cell or tissue lysate utilizing organomercury resin...
November 2018: Current Protocols in Protein Science
Vanessa Carvalho, Joachim W Pronk, Andreas H Engel
The steep increase of atomic scale structures determined by 3D cryo-electron microscopy (EM) deposited in the EMDataBank documents progress of a methodology that was frustratingly slow ten years ago. While sample vitrification on grids has been successfully used in all EM laboratories for decades, beam damage remains a road block. Developments in instrumentation and software to exploit the information carried by elastically scattered electrons made the task to achieve atomic scale resolution easier. This together with the development of fast single electron detecting cameras has resulted in unprecedented possibilities for structure determination by 3D cryo-EM...
November 2018: Current Protocols in Protein Science
Peggi M Angel, Kim Norris-Caneda, Richard R Drake
Tryptic peptide imaging is a primary workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and has led to new information reporting highly multiplexed protein localization. Technological advances within the last few years have produced robust tools for automated spraying of both matrix and enzymes. When combined with high-mass-resolution and high-mass-accuracy instrumentation, studies now generally result in two-dimensional mapping of well over 1,000 peptide peaks. This protocol describes sample preparation, spraying, and application of enzymes and matrices, and MALDI FT-ICR instrumental considerations for two-dimensional mapping of tryptic peptides from fresh-frozen or formalin-fixed, paraffin-embedded tissue sections...
November 2018: Current Protocols in Protein Science
Huiying Zhao, Ghazaleh Taherzadeh, Yaoqi Zhou, Yuedong Yang
Protein-carbohydrate interaction is essential for biological systems, and carbohydrate-binding proteins (CBPs) are important targets when designing antiviral and anticancer drugs. Due to the high cost and difficulty associated with experimental approaches, many computational methods have been developed as complementary approaches to predict CBPs or carbohydrate-binding sites. However, most of these computational methods are not publicly available. Here, we provide a comprehensive review of related studies and demonstrate our two recently developed bioinformatics methods...
November 2018: Current Protocols in Protein Science
Heungnam Kim, Mitchell Ho
Heparan sulfate (HS) plays an important role in development and disease. It interacts with many growth factors, chemokines, and other ligands known to be important for cell growth, motility, and differentiation. However, isolating an antibody to HS in mice, rabbits, or humans is difficult due to the poor immunogenicity of HS. Phage display is a major antibody engineering technology that allows the selection of antibodies for poorly immunogenic or highly conserved antigens. This protocol contains detailed procedures for HS antigen preparation and isolation of a phage displayed human single-chain Fv (HS20) that binds HS on glypican-3 (GPC3), and analysis of the selected phage antibody...
November 2018: Current Protocols in Protein Science
Sean R Gallagher
Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit composition, track post-translational modifications, and verify identity and homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration...
November 2018: Current Protocols in Protein Science
Richard R Drake, Thomas W Powers, Kim Norris-Caneda, Anand S Mehta, Peggi M Angel
Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post-translational modification. These modifications occur most commonly as one of three major classes: N-linked glycosylation on asparagine residues, O-linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N-linked glycans, an endoglycosidase enzyme, peptide N-glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar...
November 2018: Current Protocols in Protein Science
Ling Fu, Keke Liu, Renan B Ferreira, Kate S Carroll, Jing Yang
Oxidation of a protein cysteinyl thiol (Cys-SH) to S-sulfenic acid (Cys-SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S-sulfenylation, is a reversible post-translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S-sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S-sulfenylated proteins either in vitro or in situ, as it allows stabilization and direct detection of this transient oxidative intermediate...
October 12, 2018: Current Protocols in Protein Science
Molly Hunter, Ping Yuan, Divya Vavilala, Mark Fox
Recombinant proteins, such as monoclonal antibodies, are produced in mammalian cell lines to introduce proper protein folding and post-translational modifications, which are essential for full biological activity. In both the industrial and academic environments, the use of recombinant proteins varies widely and, with it, the method of production. The amount of an antibody needed for a toxicity study is far greater than that needed by a research lab performing cellular assays, and the amount of effort put into the development of the protein will vary accordingly...
September 28, 2018: Current Protocols in Protein Science
Jingjing Deng, Hediye Erdjument-Bromage, Thomas A Neubert
Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel...
September 20, 2018: Current Protocols in Protein Science
Aurora Paiano, Azzurra Margiotta, Maria De Luca, Cecilia Bucci
This article describes the general method to perform the classical two-hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein-protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two-hybrid assay have been proven to be binary direct interactions...
August 21, 2018: Current Protocols in Protein Science
Gang Hu, Lukasz Kurgan
Sequence similarity searching has become an important part of the daily routine of molecular biologists, bioinformaticians and biophysicists. With the rapidly growing sequence databanks, this computational approach is commonly applied to determine functions and structures of unannotated sequences, to investigate relationships between sequences, and to construct phylogenetic trees. We introduce arguably the most popular BLAST-based family of the sequence similarity search tools. We explain basic concepts related to the sequence alignment and demonstrate how to search the current databanks using Web site versions of BLASTP, PSI-BLAST and BLASTN...
August 13, 2018: Current Protocols in Protein Science
Kai Wen Teng, Pin Ren, Paul R Selvin
Methods to efficiently deliver fluorophores across the cell membrane are crucial for imaging the dynamics of intracellular proteins using fluorescence. Here we describe a simple protocol for permeabilizing living cells using streptolysin O, a bacterial toxin, which allows transient uptake of fluorescent probes for labeling specific intracellular proteins. The technique is applicable for delivering different classes of fluorescent probes with a molecular weight of <150 kDa, and it is also applicable to a variety of different cell lines...
August 2018: Current Protocols in Protein Science
Zoltan Nagy, Shane Comer, Albert Smolenski
Phos-tag gels are recent tools to dissect protein phosphorylation that operate by inducing a shift in the electrophoretic mobility of phosphorylated proteins compared to their nonphosphorylated counterparts. This article describes the preparation and electrophoresis of Zn2+ -Phos-tag gels along with electrotransfer of the separated phospho- and nonphosphoproteins onto a PVDF membrane using either wet-tank or semidry transfer. We also discuss the theory behind the technology with critical parameters to keep in mind for its successful application...
August 2018: Current Protocols in Protein Science
Guillaume Lenoir, Thibaud Dieudonné, Anaïs Lamy, Maylis Lejeune, José-Luis Vazquez-Ibar, Cédric Montigny
Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent...
August 2018: Current Protocols in Protein Science
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