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Current Protocols in Protein Science

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
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
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 biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development...
February 2, 2017: Current Protocols in Protein Science
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 have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved...
February 2, 2017: Current Protocols in Protein Science
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 switch or participates in a codified manner with other post-translational modifications to regulate different cellular and developmental processes in normal and pathological states. This unit describes methods for in vitro and in vivo determination of lysine acetylation...
February 2, 2017: Current Protocols in Protein Science
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 similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented...
February 2, 2017: Current Protocols in Protein Science
Matthew J Wither, Kirk C Hansen, Julie A Reisz
Recent technological advances in mass spectrometry (MS) have made possible the investigation and quantification of complex mixtures of biomolecules. The exceptional sensitivity and resolving power of today's mass spectrometers allow for the detection of proteins and peptides at low femtomole quantities; however, these attributes demand high sample purity to minimize artifacts and achieve the highest degree of biomolecule identification. Tissue preparation for proteomic studies is particularly challenging due to their heterogeneity in cell type, presence of insoluble biomaterials, and wide diversity of biomolecules...
November 1, 2016: Current Protocols in Protein Science
Fanchi Meng, Lukasz Kurgan
Secondary structure of proteins refers to local and repetitive conformations, such as α-helices and β-strands, which occur in protein structures. Computational prediction of secondary structure from protein sequences has a long history with three generations of predictive methods. This unit summarizes several recent third-generation predictors. We discuss their inputs and outputs, availability, and predictive performance and explain how to perform and interpret their predictions. We cover methods for the prediction of the 3-class secondary structure states (helix, strand, and coil) as well as the 8-class secondary structure states...
November 1, 2016: Current Protocols in Protein Science
Aaron Goldman, Sandra Harper, David W Speicher
Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics...
November 1, 2016: Current Protocols in Protein Science
Xing-Jun Cao, Benjamin A Garcia
Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites...
November 1, 2016: Current Protocols in Protein Science
Benjamin Webb, Andrej Sali
Comparative protein structure modeling predicts the three-dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and how to use the ModBase database of such models, and discusses all four steps of comparative modeling, frequently observed errors, and some applications...
November 1, 2016: Current Protocols in Protein Science
Kieran F Geoghegan
Chemical modification of amino groups in proteins serves a diversity of preparative and analytical purposes. The most prominent is to attach nonpeptide groups with useful properties to proteins. Examples of these groups include biotin for affinity capture and fluorescent dyes for detectability. A widely applied chemistry, and one for which many reagents are available, is reaction of the activated ester of a carboxylic acid (often a succinimidyl ester) with amino groups at mildly basic pH. Reductive alkylation using a carbonyl compound and a hydride-donating reducing agent is another valued reaction with multiple applications...
November 1, 2016: Current Protocols in Protein Science
(no author information available yet)
The basic theory of protein precipitation by addition of ammonium sulfate is presented, and the most common applications are listed. Tables are provided for calculating the appropriate amount of ammonium sulfate to add to a particular protein solution.
April 1, 2016: Current Protocols in Protein Science
(no author information available yet)
The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine-HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also described.
April 1, 2016: Current Protocols in Protein Science
Michael A Massiah, Katharine M Wright, Haijuan Du
This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns...
April 1, 2016: Current Protocols in Protein Science
Marcella Orwick-Rydmark, Thomas Arnold, Dirk Linke
Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy...
April 1, 2016: Current Protocols in Protein Science
Li Lei, Martin Egli
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels...
April 1, 2016: Current Protocols in Protein Science
Daumantas Matulis
Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described...
February 2, 2016: Current Protocols in Protein Science
Constance J Jeffery
Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins...
February 2, 2016: Current Protocols in Protein Science
Michal Zolkiewski, Liudmila S Chesnokova, Stephan N Witt
Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone, DnaK. The procedures for purification of these chaperones are also described.
February 2, 2016: Current Protocols in Protein Science
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