生物化学技术

Inositol(1,4,5)trisphosphate is an important second messenger that activates its cognate Ins(1,4,5)P3 receptor to release Ca2+ from intracellular stores. The assay described in this chapter uses the Ins(1,4,5)P3 receptor (essentially as a binding protein) to measure the biologic ...

Inositol 1,4,5-trisphosphate (IP3) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by enzymes of the phospholipase C (PLC) family. Binding of IP3 to its cognate receptor in the endoplasmic reticulum membrane leads to re ...

Measurement of calcium (Ca2+) fluorescence in conjunction with ionic currents is of particular importance in contractile cells, such as cardiac ventricular myocytes and vascular smooth muscle. The interplay between membrane potential and intracellular calcium (i) is fundame ...

Voltage-sensitive calcium channels (VSCC) are vital to the normal physiology of many cell types, including neurones, skeletal, cardiac and smooth muscle cells, heart pacemaker tissue and endocrine cells. Whole-cell recording is a functional electrophysiological assay that all ...

The control of free ionized intracellular calcium concentration (i) is an established mechanism of cellular activation, regulating a diverse range of cellular events. Consequentially, experimental measurement of i is a potent technique for the medical science laboratory. The NO ...

The Fluorometric Imaging Plate Reader (FLIPR) has made a significant contribution to drug discovery programs. The key advantage of FLIPR over conventional plate readers is the ability to measure fluorescence emission from multiple wells (96 wells or 384 wells) simultaneously and with ...

The FlexStation � Scanning Fluorometer is a fluorescence plate reader that can measure intracellular Ca2+ concentration using both single-wavelength and dual-wavelength fluorescent probes. The FlexStation uses a Xenon flashlamp and monochromators for both excitation and e ...

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to int ...

Use of Fura-2 in whole cell suspensions to measure changes in intracellular Ca2+ is probably one of the simplest, yet most widely used protocols described in this volume. Whole cell suspensions are loaded with Fura-2 and then placed into a cuvette-based fluorimetric system (measuring 510 nm emi ...

There is a vast array of dyes currently available for measurement of cytosolic calcium. These encompass single and dual excitation and single and dual emission probes. The choice of particular probe depends on the experimental question and the type of equipment to be used. It is therefore extre ...

Group I intron ribozymes constitute one of the main classes of ribozymes and have been a particularly important model in the discovery of key concepts in RNA biology as well as in the development of new methods. Compared to other ribozyme classes, group I intron ribozymes display considerable var ...

The principle task of the ubiquitous enzyme RNase P is the generation of mature tRNA 5′-ends by removing precursor sequences from tRNA primary transcripts (Trends Genet 19:561–569, 2003; Crit Rev Biochem Mol Biol 41:77–102, 2006; Trends Biochem Sci 31:333–341, 2006). In Bacteria, RNase P is a ribonu ...

Among the nine classes of ribozymes that have been experimentally validated to date is the metabolite-responsive self-cleaving ribozyme called glmS. This RNA is almost exclusively located in the 5′-untranslated region of bacterial mRNAs that code for the production of GlmS proteins, w ...

Group II introns are large self-splicing ribozymes found in bacterial genomes, in organelles of plants and fungi, and even in some animal organisms. Many organellar group II introns interrupt important housekeeping genes; therefore, their splicing is critical for the survival of the host ...

Detecting functional RNAs is increasingly accomplished through structure-based searches for patterns of conserved secondary structure. With large amounts of new sequencing data becoming available, there is a greater demand for efficient methods of identifying new RNAs. Here we ...

Recent developments in optical microscopy and nanometer tracking have greatly improved our understanding of cytoskeletal motor proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules mainly using a ge ...

Many single-molecule experimental techniques exploit fluorescence as a tool to investigate conformational dynamics and molecular interactions or track the movement of proteins in order to gain insight into their biological functions. A prerequisite to these experimental ap ...

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule lev ...

Cytoplasmic dynein, which is the largest and arguably the most complex cytoskeletal motor protein, plays fundamental roles during cell division, nuclear positioning, and organelle and mRNA transport, by generating force and movement toward the minus ends of microtubules. Consequ ...

In order to open the DNA double helix mechanically, a molecular construction is prepared which allows specific attachment of the two complementary strands of an individual molecule to two differentμm-sized beads. The beads are separately captured by a dual optical trap, thus holding the DNA ...