生物化学技术

UV/Vis spectroscopy is the major means of identifying intact holocytochrome P450. The carbon monoxide complex of the intact ferrous hemoprotein exhibits a characteristic spectrum between 448 and 452 nm, considerably distinct from the usual Soret absorption peaks of hemoproteins. ...

Five different transfection reagents—calcium phosphate, TransFast™ Transfection Reagent, Superfect™ Transfection Reagent, Effectene™ Transfection Reagent, and Tfx™-20—were compared for their ability to effectively transfect primary cultures of male rat hepatocy ...

Stem cell research has become an important field of study for molecular, cellular, and clinical biology as well as pharmaco-toxicology. Indeed, stem cells have a strong proliferative and unlimited self-renewal potential and are multipotent. In vivo as well as in vitro studies have confirm ...

Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocyt ...

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from F ...

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technolo ...

Transcriptional activation of CYP gene expression by xenobiotics may have fundamental effects on body physiology. It may result in the altered pharmacokinetics of other chemicals in the body, both xenobiotic and endogenous substrates, potentially altering their effects. This may ...

Cytochromes P450 (CYP) have been expressed in a variety of systems such as mammalian cells, yeast, and bacteria. The bacterial system is technically the least demanding and provides large amounts of catalytically active P450s for metabolic and structural studies relating to preclinic ...

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mi ...

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have ...

Human liver microsomes contain multiple forms of cytochrome P450 (CYP or P450) that catalyze oxidation of a number of xenobiotic and endobiotic chemicals. Individual P450 forms have unique, but overlapping, substrate specificities. It is necessary to determine which P450s play more i ...

Recent developments in recombinant DNA technology have enabled the synthesis of valuable therapeutic proteins in bacterial cells as well as in novel eucaryotic expression systems. However, the purification of proteins of interest from either the conventional sources, cell cult ...

Biopolymers can be separated by partitioning between two aqueous phases generated by two polymers dissolved together in water (1,2). The partitioning of proteins and nucleic acids between the two phases may be affected by changing the concentration of polymers, usually dextran and poly ...

The use of boronate affinity chromatography for separation of nucleic acid components and carbohydrates was first reported by Weith and colleagues in 1970 (1). Since then, the specificity of boronate has been exploited for the separation of a wide variety of cis-diol-containing compoun ...

Affinity chromatography has proven to be the most effective technique for the purification and separation of proteins from complex mixtures (1). Although affinity adsorbents based on biological ligands such as immobilized antibodies, lectins and nucleotide cofactors appear to ...

Exogenous pyrogens originating in the cell wall of Gram-negative bacteria have the strongest pyrogenicity (1), and for them synonyms such as endotoxins, lipopolysaccharides (LPS), and O-antigens are used. A number of attempts using affinity chromatographic approaches have been ma ...

DNA affinity chromatography has been used for the purification of polynucleotides and polynucleotide-binding proteins, including restriction endonucleases, polymerases, proteins involved in recombination, and various transcription factors (reviewed in ). The earl ...

The reader may be surprised to find a chapter about a Western Blotting technique in a book dealing with affinity chromatography. However, the method described here combines two steps that are characteristic of immuno affinity chromatography. One is the preparation of monospecific anti ...

Applications of affinity chromatography for quantitative analysis (1–10), purification at laboratory scale (11–16), and for large-scale manufacture of recombinant DNA technology derived therapeutics (17–23), have been continually expanding since the first application i ...

The need to replace natural amino acids in peptides with nonproteinogenic counterparts in order to obtain drug-like target molecules has stimulated a great deal of innovation on several fronts (1–3). One of the more exciting areas of research in drug design has been the synthesis of so-called se ...