生物化学技术

Cyclic AMP governs many fundamental signaling events in eukaryotic cells. Although cAMP signaling has been a major research focus for a long time, recent technological developments are revealing novel aspects of this paradigmatic pathway. In this chapter, we give an overview over curre ...

Monitoring cellular calcium concentration using fluorescent reporters can provide a rapid, proportional assay of G-protein-coupled receptor activation. Recording calcium changes in single cells, or cell populations, is relatively straightforward, but requires caref ...

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through �promoting guanine nucleotide exchange on Gα subunits of heterotrimeric G proteins to release Gα-GTP and free βγ-subunits. In membrane preparations, GTP can be substituted by 35S-lab ...

The radioligand binding assay is a relatively simple but powerful tool for studying G protein-coupled receptors. There are three basic types of radioligand binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site d ...

Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain r ...

In this chapter, we describe a method for detecting the ubiquitination status of G protein-coupled receptors (GPCRs). This involves co-expression of a GPCR with an epitope-tagged ubiquitin construct in a �heterologous mammalian expression system. Stimulus-dependent modifica ...

Phosphorylation of G protein-coupled receptors (GPCRs) is one of the most prominent post-translation modifications mediated by agonist stimulation. This process has been shown to result not only in receptor desensitisation but also, via the recruitment of arrestin adaptor prote ...

The drive to produce safer and more receptor subtype selective drugs has sparked a renewed interest in allosteric modulators of G protein-coupled receptors. The increasing use of functional assays has shown that allosteric ligands are capable of modulating both orthosteric agonist a ...

In this chapter, we describe methods to heterologously express G protein-coupled receptors (GPCRs) in the fission yeast Schizosaccharomyces (Sz.) pombe. GPCRs regulate a diverse range of biological processes in all eukaryotic cells, including plants, insects, humans, and yeast. The ...

The ability to assess whether individual proteins are involved in the signalling or regulation of G �protein-coupled receptor signalling is highly dependent on the pharmacological tools available. In the absence of appropriate pharmacological agents, alternative molecular ...

The first tandem affinity purification (TAP) protocol was described in 1999. Originally designed for the purification of protein complexes in yeast RNA splicing, its application rapidly expanded towards whole proteome analysis in yeast and mammalian cells. More recently, TAP has be ...

Localization and trafficking of G protein-coupled receptors (GPCRs) is increasingly recognized to play a fundamental role in receptor-mediated signaling and its regulation. Individual receptors, including closely homologous subtypes with otherwise similar function ...

Statistical methods appropriate in research are described with examples. Topics covered include the choice of appropriate averages and measures of dispersion to summarize data sets, and the choice of tests of significance, including t-tests and a one- and a two-way ANOVA plus post-tests f ...

Biochemical or pharmacological studies of G protein-coupled receptors (GPCRs) are widely conducted in transfected mammalian cells. A variety of commercially available systems allow the generation of stable cell-lines in which expression of the recombinant receptor can be indu ...

Lipid domains of the plasma membrane were originally described as a cell matrix insoluble in cold �nonionic detergents and enriched in glycosphingolipids. Because of these biochemical properties, these membrane domains were termed detergent-resistant membranes (DRMs) or det ...

Tissue microarray (TMA) is a highly efficient method that allows for large-scale measurement of �expression of RNA or protein in multiple tissue sections simultaneously. Most TMAs are made from paraffin-�embedded tissues. In this chapter, we detail a method that enables construction of ...

Immunohistochemistry (IHC) is the gold standard methodology for in-situ protein expression analysis in tissue samples. The combination of IHC and tissue microarray (TMA) technology allows for the simultaneous analysis of hundreds of tissue samples with an unprecedented degree of ...

Although most tissue microarray (TMA) slides are currently made from paraffin-embedded tissues, �frozen clinical tissues are also gradually being used to prepare TMAs. This is because frozen tissues contain better quality RNAs and proteins for profiling gene expressions. Here, we in ...

The construction of tissue microarrays from needle core biopsy specimens allows for the study of diseases with limited tissue samples and provides insight into tumors that are treated with nonsurgical approaches. While the techniques are technically challenging, in the hands of inve ...

Tissue microarray technology is a new method used to analyze 100s to 1,000s of tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing t ...