Current Protocols Essential Laboratory Techniques最新文献

Chromatography in its many different forms is an indispensable separation technique for purification or analysis of one or multiple components from a mixture that may contain a variety of simple as well as complex molecules. This unit is focused on a specific type of chromatography known as liquid chromatography (LC), with particular reference to separation of proteins. The theory of chromatography is discussed in the context of partition of the components of a mixture between a solid stationary phase and a liquid mobile phase. Although very useful for chromatographic separation of small molecules, considerable deviation is observed in the case of separation of proteins. The principles of three commonly used chromatographic techniques for separation of proteins, such as size exclusion, ion exchange, and affinity chromatography, are discussed. The unit is expected to be useful for biologists and biochemists, as well as engineers involved in downstream bioprocessing. © 2016 by John Wiley & Sons, Inc.

Next-generation sequencing (NGS) technologies have revolutionized the biosciences and become invaluable to the discovery of gene function and its involvement in disease conditions. The fast pace of innovation in NGS technologies has enabled the production of huge volumes of sequence data at progressively lower cost. However, the increasing throughput combined with the growing accessibility of these technologies poses significant challenges for downstream data analysis. Here, we provide an overview of NGS methods and key secondary analysis pipelines with a focus on technology provided by Illumina. As a case study, we highlight potential applications in cancer research. © 2016 by John Wiley & Sons, Inc.

Immunoblotting (also referred to as western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. Specialized blotting equipment electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression, and post-translational modification analysis. © 2016 by John Wiley & Sons, Inc.

In the post-genomic era, more and more research projects involve the generation of molecular sequence data. How should these newly obtained DNA/protein sequences be analyzed, and how should they be prepared for submission to sequence databases? In this unit, we provide guidelines and a flowchart to help first-time users process new sequence data using third-party freeware programs and give a step-by-step demonstration on the preparation of a sequence file for submission to GenBank using the Sequin program. © 2016 by John Wiley & Sons, Inc.

In this molecular day and age, microscopy seems to be a neglected field of instruction. Too often professors, who themselves are strangers to the use of the light microscope, may hurry through a laboratory exercise designed to familiarize students with its uses. This chapter is designed to serve as a useful reference for instructors, perhaps demystifying some features of the microscope, and making their applications more user-friendly and exciting. © 2016 by John Wiley & Sons, Inc.

Spectroscopy is the study of the interaction of light with matter. By observing how light interacts with matter—through reflection, refraction, elastic scattering, absorbance, inelastic scattering, and emission—it is possible to (1) identify the wavelengths of light that interact with atoms and molecules, and (2) quantify the amount of light being absorbed, reflected, scattered, or emitted at a particular wavelength. This unit describes the background and basic principles of spectrophotometry (the study of the reflection or transmission properties of a substance as a function of wavelength), in particular, absorbance spectrophotometry. Also included is discussion of key spectrophotometer components and their functions, the relationship between absorbance and transmittance, experimental considerations, and the steps necessary in preparing a standard curve for determining absorbance concentration. © 2015 by John Wiley & Sons, Inc.

The ubiquitous use of digital imaging has greatly simplified and accelerated life science research. However, the ease of manipulating digital images also presents serious ethical issues. This unit presents guidelines and cautions describing best practices for use, analysis, and presentation of digital image data. © 2015 by John Wiley & Sons, Inc.

In recent times, varieties of modified synthetic oligonucleotides conjugated with reporter molecules have been employed in hybridization-based assays. These methods have broad applications in both basic molecular biology research and in clinical diagnostics and screening because they have high specificity to a target nucleic acid sequence. The development of the fluorescence resonance energy transfer (FRET) process has improved the performance and utility of these hybridization-based assays. In this unit, we discuss the main principles of the design and synthesis of the qPCR probes. © 2015 by John Wiley & Sons, Inc.

This unit provides information to aid in the selection and proper use of a laboratory balance. Laboratory balances are used for measuring the mass of an object and come in two main types: mechanical and electronic. Electronic balances generally come with a computer interface to facilitate the collection, storage, and manipulation of the data. In addition to weighing objects, electronic balances also perform a range of computations, including counting objects, measuring density, statistics, and pipet volume calibration. Balances vary widely in terms of their capacity (how heavy an object they can accurately weigh), precision, accuracy, repeatability, and robustness. Understanding the various characteristics of a laboratory balance is necessary in order to be sure that the balance is well suited for your particular scientific or industrial needs. This unit also discusses the proper use and maintenance of a laboratory balance. In general, laboratory balances are relatively easy to use and require little maintenance. © 2015 by John Wiley & Sons, Inc.

Fluorescence is an extremely powerful tool in modern biology, physics, and chemistry laboratories. This unit begins with the physics of fluorescence, the biological applications of fluorescence, and the mechanisms behind spectrometers and fluorometers, followed by strategies to choose an appropriate fluorophore according to the instrumentation at hand. The first part of the unit walks the reader through the acquisition of an absorption spectrum and explains how it can be used to measure the degree of labeling on conjugated proteins. The discussion then extends into how to obtain fluorescence emission spectra, which can be utilized as fluorescence indicators for protein detection. © 2015 by John Wiley & Sons, Inc.