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Methods in Enzymology

Richard B Thompson, Carol A Fierke
No abstract text is available yet for this article.
2017: Methods in Enzymology
Remco Arts, Stijn J A Aper, Maarten Merkx
FRET-sensors have become important tools for intracellular imaging, but their dependence on external illumination presents some limitations, such as photobleaching and phototoxicity, which limit measurements over extended periods of time. Fluorescence measurements also suffer from autofluorescence and light scattering, which hampers in vivo imaging and measurements in strongly absorbing and scattering media such as blood. In principle, these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET)...
2017: Methods in Enzymology
Daniel Wynn, Sapna Deo, Sylvia Daunert
Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited...
2017: Methods in Enzymology
Huai Deng, Tom K Kerppola
We have developed a procedure that enables visualization of the genomic loci that are bound by complexes formed by a specific combination of chromatin-binding proteins. This procedure is based on imaging bimolecular fluorescence complementation (BiFC) complexes on Drosophila polytene chromosomes. BiFC complexes are formed by the facilitated association of two fluorescent protein fragments that are fused to proteins that interact with, or are in close proximity to, each other. The intensity of BiFC complex fluorescence at individual genomic loci is greatly enhanced by the parallel alignment of hundreds of chromatids within the polytene chromosomes...
2017: Methods in Enzymology
Anthony E G Cass, Sanjiv Sharma
Microneedle enzyme sensors by virtue of their minimally invasive and hence pain-free penetration of skin allow for the measurement of metabolites, biomarkers, and drugs in the interstitial fluid that bathes the dermal tissue. Such devices if they are to be adopted widely into clinical practice need to be capable of delivering reliable measurements over extended periods of time (days) and to be fabricated by low-cost, scalable methods. Using injection molding of the base structures in polycarbonate, metal film deposition by sputtering and enzyme immobilization by electrodeposition can meet these requirements...
2017: Methods in Enzymology
Caitlin E Anderson, Kamal G Shah, Paul Yager
The design of appropriate diagnostic assays for the point of care requires development of suitable biosensors, detection methods, and diagnostic platforms for sensitive, quantitative detection of biological analytes. Protein targets in particular are especially challenging to detect quantitatively and sensitively due to the lack of amplification strategies akin to nucleic acid amplification. However, recent advances in transducer and biosensor design, new detection labels, and paper-based microfluidics may realize the goal of sensitive, fast, portable, and low-cost protein detection...
2017: Methods in Enzymology
Qiuliyang Yu, Rudolf Griss, Alberto Schena, Kai Johnsson
Obtaining patient-specific information through the quantification of small molecules and proteins in bodily fluids is essential for personalized therapies. Point-of-care (POC) diagnostic devices hold the promise of delivering such benefit to a wide range of patients. However, there is a lack of enabling technology, as the majority of newly developed POC devices focus on the same underlying core technologies. Here we provide an overview of a new technology based on highly modular bioluminescent sensors that enables the quantification of small molecules and proteins at the POC with low-cost devices...
2017: Methods in Enzymology
Bruce R Branchini, Tara L Southworth
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP...
2017: Methods in Enzymology
Eduard Dumitrescu, Silvana Andreescu
Recent progress in the electrochemical field enabled development of miniaturized sensing devices that can be used in biological settings to obtain fundamental and practical biochemically relevant information on physiology, metabolism, and disease states in living systems. Electrochemical sensors and biosensors have demonstrated potential for rapid, real-time measurements of biologically relevant molecules. This chapter provides an overview of the most recent advances in the development of miniaturized sensors for biological investigations in living systems, with focus on the detection of neurotransmitters and oxidative stress markers...
2017: Methods in Enzymology
Richard B Thompson, Carol A Fierke
Fluorescence-based biosensors have shown themselves to be a powerful tool for the study of a variety of chemical species in biological systems, notably including metal ions. This chapter provides an overview of several important issues in using such sensors to study metallobiochemistry. These issues include selectivity for the analyte over potential interferents, including those that do not themselves induce a signal, the different forms in which metal ions are found (speciation), the utility of metal ion buffers, and the importance of kinetics in studying metal ion binding reactions...
2017: Methods in Enzymology
Kei Yamaura, Shigeki Kiyonaka, Itaru Hamachi
Protein-based fluorescent biosensors are powerful tools for quantitative detection of biomolecules or drugs with high sensitivity under physiological conditions. However, conventional methods for construction of biosensors require structural data with high resolution or amino acid sequence information in most cases, which hampers applicability of this method to structurally complicated receptor proteins. To sidestep such limitations, we recently developed a new method that employs ligand-directed chemistry coupled with a bimolecular fluorescence quenching and recovery system, which enabled the conversion of various kinds of membrane-bound receptors to "turn-on" type fluorescent sensors...
2017: Methods in Enzymology
Rebecca Y Lai
A number of electrochemical DNA sensors based on the target-induced change in the conformation and/or flexibility of surface-bound oligonucleotides have been developed in recent years. These sensors, which are often termed E-DNA sensors, are comprised of an oligonucleotide probe modified with a redox label (e.g., methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of the target to the DNA probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode...
2017: Methods in Enzymology
Wei Niu, Jiantao Guo
Fluorescent proteins of different colors are useful probes to study protein structure and function, and to investigate cellular events and conditions. Large efforts have focused on engineering new properties into fluorescent proteins via rational design and directed evolution. In addition to applications in imaging of protein expression level and subcellular localization, fluorescent proteins have been increasingly engineered to act as biosensors to track concentrations of small-molecule metabolites, enzyme activities, and protein conformational changes in living cells...
2017: Methods in Enzymology
Satoshi Okada, Mid Eum Lee, Erfei Bi, Hay-Oak Park
Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins...
2017: Methods in Enzymology
Robert H Newman, Jin Zhang
In order to survive and function properly in the face of an ever changing environment, cells must be able to sense changes in their surroundings and respond accordingly. Cells process information about their environment through complex signaling networks composed of many discrete signaling molecules. Individual pathways within these networks are often tightly integrated and highly dynamic, allowing cells to respond to a given stimulus (or, as is typically the case under physiological conditions, a combination of stimuli) in a specific and appropriate manner...
2017: Methods in Enzymology
Maria Staiano, Angela Pennacchio, Antonio Varriale, Alessandro Capo, Adelia Majoli, Clotilde Capacchione, Sabato D'Auria
Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future...
2017: Methods in Enzymology
Lynn Sanford, Amy Palmer
Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, fluorescence-modulated single FP-based sensors, translocation sensors, complementation sensors, and dimerization-based sensors. We discuss elements of sensor design and engineering for each platform, including the incorporation of new types of FPs and sensor screening techniques...
2017: Methods in Enzymology
L Galluzzi, J M Bravo-San Pedro, G Kroemer
No abstract text is available yet for this article.
2017: Methods in Enzymology
Y Zheng, Y Qiu, J E C Gunderson, B A Schulman
Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins...
2017: Methods in Enzymology
I Tanida, T Ueno, Y Uchiyama
In this chapter, we introduce the usage of pHluorin-mKate2-human LC3 for monitoring autophagy. Using EGFP and RFP, tandem fluorescent protein-tagged LC3 has been generated for monitoring autophagic structures. A critical point for this purpose is the sensitivity of the green fluorescent protein to acidic pH. A super-ecliptic pHluorin is most sensitive to acidic pH among EGFP, mWasabi, and pHluorin, indicating pHluorin is most suitable for monitoring autophagic structures. During autophagy, green-positive and red-positive fluorescent puncta of pHluorin-mKate2-human LC3 indicate signals of preautophagosomes and autophagosomes...
2017: Methods in Enzymology
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