生物化学技术

The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that plays a major role in intracellular protein degradation. In mammalian cells, this symmetrical cylindrical complex is composed of two copies each of seven different α and β subunits arranged into f ...

Exosomes are small natural membrane vesicles released by a wide variety of cell types into the extracellular compartment by exocytosis. The biological functions of exosomes are only slowly unveiled, but it is clear that they serve to remove unnecessary cellular proteins (e.g., during ret ...

Glycoproteins constitute a large fraction of the proteome. The fundamental role of protein glycosylation in cellular development, growth, and differentiation, tissue development, and in host–pathogen interactions is by now widely accepted. Proteome–wide characterizati ...

We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptidecentric proteomics approach using the principle of diagonal chromatography. We will indicate that the ...

In the present chapter we detail how mass spectrometry (MS) can be used to characterize noncovalent complexes, especially multimeric proteins and protein/ligand complexes. This original application of MS, also called “supramolecular MS” or “nondenaturing MS,” appeared in the early ...

Protein arrays make possible the functional screening of large numbers of immobilized proteins in parallel. To facilitate the supply of proteins and to avoid their deterioration on storage, we describe our protein in situ array (PISA) method for production of protein arrays in a single step d ...

Ribosome display is a powerful method for selection of single-chain antibodies in vitro. It operates through the formation of libraries of antibody–ribosome–mRNA complexes that are selected on immobilized antigen, followed by recovery of the genetic information from the mRNA by RT-P ...

Signaling pathways transduce extracellular stimuli from the membrane to the nucleus. Constitutive and thus inappropriate stimulation of these kinase cascades is associated with and observed in a majority of tumors. The transduction of signals in these pathways is achieved through ...

Networks of interacting protein control physiological processes in all living cells. Considerable effort has recently been invested in understanding protein interactions under normal and diseased conditions. One approach to elucidate the composition of protein complexes ...

In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for det ...

One of the more important new insights in the field of molecular biology in the past 15 years was the discovery that RNA molecules, once thought to be primarily passive carriers of genetic information, can carry out some functions previously ascribed to proteins. Some RNAs are capable of acting as en ...

The hairpin ribozyme has been developed and designed to target and cleave efficiently heterologous RNA sequences in a trans-reaction (1–3). Heterologous RNA cleavage has been carried out, targeting rules defined, systems identified, and very effective in vivo downregulation of gene ...

The development of capillary electrophoresis (CE) technology has been rapid over the past three years for application to the analytical separation in a variety of biopoly-mers such as proteins, polysaccharides, and DNA (1–3). CE offers high throughput and high resolution, automatic ope ...

In comparison to the sequencing of other biological macromolecules such as nucleic acids or proteins, the sequencing of oligosaccharides (glycans) of glycoproteins generally requires specialized expertise and facilities Whereas techniques such as nuclear magnetic reson ...

Recent advances in the mechanisms of regulation of leukotriene and prostaglandin biosynthesis, as well as their mechanisms of action, have generated a renewed interest in this field. One can cite the cloning of enzymes involved in the lipoxygenase (LOX) and cyclo-oxygenase (COX or PGHS) pat ...

Certain analogs of the extracellular matrix (ECM) have been shown to possess surprising morphogenetic activity during healing of lesions in various anatomical sites. This chapter describes methods for synthesis of the two ECM analogs that have been studied most extensively. The read ...

Cannabis sativa L. and its products comprise a significant and important part of the forensic drug laboratory’s case load. Two principle types of analyses are required for the analysis of Cannabis, namely, identification of the material, since it is a substance controlled in the United Kingdom ...

Rapid lead discovery in the drug development process relies on highthroughput screening of diverse compounds against protein targets. These compounds come from traditional organic synthesis, natural products isolation, and combinatorial synthesis (1). For compounds from c ...

Much of the excitement over nitric oxide (NO) is owing to its diverse physiologic and pathophysiologic functions. Induced NO production has been shown to have both beneficial and detrimental consequences. The inducible or high-output NO synthase (NOS) pathway was first characterized ...

A blessing and curse of capillary electrophoresis (CE) is the small amount of material that can be injected into the column (1–4). If one has a tiny quantity of substance to analyze, volumes on the order of 2–10 nL can be injected into a capillary column with good reproducibility; thus, precious material is n ...