Read by QxMD icon Read

Methods: a Companion to Methods in Enzymology

Annette C Moser, Sidney Trenhaile, Kati Frankenberg
Antibody-antigen interactions are vital in immunoassay development and can determine detection limits and analysis times. Capillary electrophoresis (CE) is a powerful technique that can be used to quantify antibody-antigen interactions. These CE methods range from simple separations of a premixed antibody and antigen sample applied as a short plug to allow for separation of complex, free antibody, and free antigen to more complex systems which inject complexed samples in the presence of antibody or antigen; or even injections of antibody and antigen sequentially...
March 15, 2018: Methods: a Companion to Methods in Enzymology
Sijin Guo, Xijun Piao, Hui Li, Peixuan Guo
The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNA nanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications have been extensively explored. Now it is time to offer different levels of RNA construction methods for both the beginners and the experienced researchers or enterprisers. The first and second parts of this article will provide instructions on basic and simple methods for the assembly and characterization of RNA nanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA packaging motor...
March 9, 2018: Methods: a Companion to Methods in Enzymology
Weixiang Jin, M Fethullah Simsek, Arnd Pralle
It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusiong of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components...
March 9, 2018: Methods: a Companion to Methods in Enzymology
Jacqueline Maher, Marcus Allen
There are a number of methods of investigating the function of recombinant proteins such as ion channels. However, after channel purification there are few methods to guarantee that the protein still functions. For ion channels, reconstituting back into planar lipid bilayers and demonstrating preserved function is a convenient and trusted method. It is cell free and even inaccessible, intracellular ion channels can be studied. We have used this method to study the function of recombinant channels of known subunit composition and have found it convenient for investigating the mode of action of ion channel modulators...
March 8, 2018: Methods: a Companion to Methods in Enzymology
Matthew A Zambrello, Adam D Schuyler, Mark W Maciejewski, Frank Delaglio, Irina Bezsonova, Jeffrey C Hoch
The development of multidimensional NMR spectroscopy enabled an explosion of structural and dynamical investigations on proteins and other biomacromolecules. Practical limitations on data sampling, based on the Jeener paradigm of parametric sampling of indirect time domains, have long placed limits on resolution in the corresponding frequency dimensions. The emergence of nonuniform sampling (NUS) in indirect time dimensions circumvents those limitations, affording high resolution spectra from short data records collected in practically realized measurement times...
March 6, 2018: Methods: a Companion to Methods in Enzymology
Carlo Annunziatella, Andrea M Chiariello, Andrea Esposito, Simona Bianco, Luca Fiorillo, Mario Nicodemi
In recent years interest has grown on the applications of polymer physics to model chromatin folding in order to try to make sense of the complexity of experimental data emerging from new technologies such as Hi-C or GAM, in a principled way. Here we review the methods employed to efficiently implement Molecular Dynamics computer simulations of polymer models, focusing in particular on the String&Binders Switch (SBS) model. The constant improvement of such methods and computer power is returning increasingly more accurate insights on the structure and molecular mechanisms underlying the spatial organization of chromosomes in the cell nucleus...
March 6, 2018: Methods: a Companion to Methods in Enzymology
Shaunak Kar, Andrew D Ellington
T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. Furthermore, the fact that T7 RNAP is largely orthogonal to most hosts enables its use in a wide variety of contexts. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions with and impact on physiology, and potentially leading to mutations to constructs...
March 5, 2018: Methods: a Companion to Methods in Enzymology
Roxanne Oshidari, Karim Mekhail
The health of an organism is intimately linked to its ability to repair damaged DNA. Importantly, DNA repair processes are highly dynamic. This highlights the necessity of characterizing DNA repair in live cells. Advanced genome editing and imaging approaches allow us to visualize damaged DNA and its associated factors in real time. Here, we summarize both established and recent methods that are used to induce DNA damage and visualize damaged DNA and its repair in live cells.
March 5, 2018: Methods: a Companion to Methods in Enzymology
Sandya R Beeram, Xiwei Zheng, Kyungah Suh, David S Hage
A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents...
March 3, 2018: Methods: a Companion to Methods in Enzymology
Romez Uddin, John Simms, David Poyner
Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET)...
March 3, 2018: Methods: a Companion to Methods in Enzymology
Janin Glaenzer, Martin F Peter, Gregor Hagelueken
In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility...
March 3, 2018: Methods: a Companion to Methods in Enzymology
Antonio N Calabrese, Sheena E Radford
The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins...
March 3, 2018: Methods: a Companion to Methods in Enzymology
Leonel Malacrida, Estella Rao, Enrico Gratton
Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the cell. The 2D-pCF does not require spatial averaging and it gives information about obstacles for diffusion at pixel resolution. We show the analysis of the same set of data by the two methods to emphasize that both methods could be needed to have a comprehensive understanding of the molecular diffusional flow in a live cell...
February 28, 2018: Methods: a Companion to Methods in Enzymology
Silvia Kocanova, Isabelle Goiffon, Kerstin Bystricky
Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35kb to 300kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains...
February 28, 2018: Methods: a Companion to Methods in Enzymology
Srikanth Gattu, Cassandra L Crihfield, Grace Lu, Lloyd Bwanali, Lindsay Veltri, Lisa A Holland
Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations...
February 27, 2018: Methods: a Companion to Methods in Enzymology
Colin S Maxwell, Thomas Jacobsen, Ryan Marshall, Vincent Noireaux, Chase L Beisel
The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases...
February 24, 2018: Methods: a Companion to Methods in Enzymology
David Hardy, Elodie Desuzinges Mandon, Alice Rothnie, Anass Jawhari
Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation...
February 22, 2018: Methods: a Companion to Methods in Enzymology
Chia-Hsiu Chen, Yunyi Wang, Christian Hilty
The nuclear Overhauser effect (NOE) is a primary means to characterize intermolecular interactions using modern NMR spectroscopy. Multiple experiments measured using different mixing time can be used for quantifying NOE buildup and measuring cross-relaxation rates. However, this approach using conventional multi-dimensional NMR is time consuming. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) can generate deviations from equilibrium spin polarization by orders of magnitude, thereby enhancing signals and allowing to characterize NOE build up in real-time...
February 19, 2018: Methods: a Companion to Methods in Enzymology
Caterina Temporini, Gloria Brusotti, Giorgio Pochetti, Gabriella Massolini, Enrica Calleri
Affinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. Pros and cons of the approaches in ligand discovery initiatives...
February 17, 2018: Methods: a Companion to Methods in Enzymology
Johnny Truong, Yu-Fang Hsieh, Lynda Truong, Guifang Jia, Ming C Hammond
RNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology...
February 16, 2018: Methods: a Companion to Methods in Enzymology
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"