生物化学技术

Complex genomes are characterized by large amounts of tandemly repeated DNA that can comprise up to several percent of the genome in some organisms (1,2). The analysis of the organization of this type of DNA presents certain challenges owing to its repetitive nature, genomic distribution, and ...

Pulsed-field gel electrophoresis (PFGE) has the capacity to fractionate large fragments of DNA up to thousands of kilobases in size. This aspect of the technique has been exploited for constructing long-range restriction maps of chromosomes from many different species including hum ...

The introduction of pulsed-field agarose gel electrophoresis (PFGE) has expanded the list of particles separable by use of gel electrophoresis to include: (1) linear DNAs as long as 3–6 Mbp, (2) DNA- protein complexes and circular DNAs that become arrested during invariant field agarose gel el ...

Pulsed-field gel electrophoresis (PFGE) is one of the key technological advances of the past ten years that has made the mapping of genomes of whole organisms possible. In conventional electrophoreis, the mobility of DNA at almost any practical value of the field strength is essentially inde ...

DNA molecules less than ~20 kilobase (kb) pairs in size are efficiently fractionated by unidirectional electrophoresis in agarose gels (1). The variation of DNA mobility with gel concentration can be described by the Ogston mechanism of pore size distribution, if the observed mobilities ...

Gel electrophoresis of DNA has improved greatly in the last decade, mostly owing to the invention of pulsed-field gel electrophoresis (1). This has enabled the separation of DNA more than two orders of magnitude longer than what was previously possible (2–4). This chapter will review what is unde ...

In the history of the still-young technique of pulsed electrophoresis experimental, theoretical, and numerical progress are increasingly intermingled. The first key to this field can be traced to theoretical letters of Lerman and Frisch and Lumpkin and Zimm (1,2), who remarked that long f ...

Gel electrophoresis is one of the most common techniques used in molecular biology for the separation of DNA molecules. Conventional gel electrophoresis (using a static electric field) does not permit separation of DNA fragments larger than 30–50 kbp (1) as shown in Fig. 1A of Chapter 7. This is a surp ...

Probing ion channel structure-function and regulation in native tissue can, in some instances, be experimentally challenging or impractical. To facilitate discovery and increase experimental flexibility, our laboratory routinely reconstitutes recombinant ion chann ...

Overexpression of proteins is a powerful way to determine their function. Until recently, the low efficiency of neuronal transfection has made it difficult to use overexpression and structure-function studies to investigate the role of neuronal proteins in their native environme ...

The amiloride-sensitive epithelial Na+ channel (ENaC) is typically composed of three structurally related subunits termed α, β, and γ. We describe methods to determine the functional subunit stoichiometry of ENaC based on a biophysical approach that was first introduced in 1991 to deter ...

Voltage-gated potassium channels are ubiquitous and critical for life. They must fold and assemble correctly and target to appropriate sites in the plasma membrane. Failure to do so can lead to inappropriate targeting or function and to pathology. The methods described here were developed ...

Exogenous expression of genes in mammalian neurons represents a substantial experimental challenge because of the low efficiency of commercially available liposomal transfection reagents for nondividing cells and considerable toxicity of viral transfection systems. ...

The combination of green fluorescent protein mutants and fluorescence resonance energy transfer (FRET) forms a powerful tool for ion channel studies. A key to successful application of green fluorescent protein-based FRET is to reliably separate the FRET signal from various non-FRET ...

Acute brain slices allow electrophysiological and imaging techniques to be applied vitro to the study of neuronal ion channels, synaptic plasticity, and whole-cell function in juvenile and adult tissue. Ion channel recordings from small dendritic branches axons in brain slices have d ...

Extracellular recording of the action potential discharge of individual neurons has been an indispensable electrophysiological method for more than 50 yr. Although it requires relatively modest instrumentation, extracellular recording nevertheless provides critic ...

Activation of Ca2+ channels in the plasma membrane or on internal Ca2+ stores raises cytosolic Ca2+ concentration (c). Among diverse functions of Ca2+ signals, the induction of exocytosis—the process in which the contents of secretory vesicles are released by their fusion to the plasma membr ...

Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. Ho ...

Amiloride-sensitive Na+ channels belong to the epithelial Na+ channel (ENaC)-degenerin superfamily of ion channels. In addition to their key role in sodium handling, they serve diverse functions in many tissues. Improper functioning of ENaC has been implicated in several diseases, in ...

Ion channels are integral membrane proteins that control transmembrane ion fluxes to regulate membrane potential, cell excitability, and ion transport. Membrane phospho-lipids containing phosphoinositides have recently emerged as important regulators of many ion chann ...