生物化学技术

The phosphoinositide cascade plays a central role in the transduction of many extracellular stimuli. Activation of plasma membrane-associated phosphoinositide-specific phospholipase C stimulates the hydrolysis of phosphatidylinositol-4,5-bisphosphate (Ptd ...

Any chromatographic procedure for the separation of phosphoinositols will require a supply of standards, both for validation of the technique and identification of the products. Tritium-radiolabeled standards are commercially available in 1–2 μCi quantities for Ins(1,3,4,5) ...

With the discovery of the role of polyphosphoinositide hydrolysis in mediating the action of calcium-mobilizing hormones, growth factors, and neurotransmitters (reviewed in ref. 1), the development of methods for measuring the formation of the resultant second messengers, inos ...

Phosphoinositide metabolism plays a central role in the transduction of signals triggered by a wide variety of growth factors and hormones. Phosphoinositides have been implicated in cellular processes as diverse as proliferation and differentiation, cytoskeletal rearrang ...

Solubilization and accurate quantification of cellular protein are central to analytical methods such as Western analysis. Alternate homogenization buffers can be used according to need, but must match the assay used. The Bio-Rad (Hercules, CA) assay, based on the method of Bradford (1), is ...

The phosphatidylinositol 3-kinases (PI 3-kinases), which play a pivotal role in cellular signaling, represent a family of enzymes that phosphorylate phosphoinositides on the 3-position of the inositol ring (1). PtdIns(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate) ...

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1) combined with Western immunoblot analysis (2,3) is a widely used technique for quantitatively and qualitatively evaluating specific protein expression. This chapter describes a Western immunob ...

Immunohistochemistry and immunocytochemistry are powerful techniques for localizing the molecular expression of proteins in tissues and cells, especially when combined with the in situ hybridization technique. Immunohistochemistry and immunocytochemistry tech ...

Total undegraded cellular RNA, largely free of contaminating DNA, can be rapidly and easily isolated from homogenized tissue or cultured cells using acid pH-guanidinium thiocyanate/phenol/chloroform extraction (1). Commercially available acid phenol/guanidinium thio ...

Because there are several nuclear substrates for the members of the mitogen-activated protein kinase (MAPK) superfamily, they should become localized to the nucleus to achieve their functions. For example, whereas extracellular signal-regulated kinase (ERK) predominantly l ...

The p42 and p44 mitogen-activated protein kinases (MAPKs) (p42/p44 MAPK, Erk2 /Erk1) are activated through the small G protein Ras and sequential activation of the protein kinases Raf and MEK on stimulation of cells with a broad range of extracellular signals (1,2). This signaling pathway, con ...

We predict that computational modeling platforms will soon become standard tools in experimental laboratories involved in the study of complex regulatory networks of various cellular processes—a field of research where an avalanche of genomic, proteomic and other biochemical d ...

Growth factor receptors (GFRs) represent a subset of a large class of proteins that serve as extracellular environment sensors of the cell. They are single transmembrane proteins with an extracellular ligand-binding domain (LBD) and an intracellular tyrosine kinase domain (1,2). Ind ...

G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface molecules involved in signal transmission. These receptors play key physiologic roles, with their dysfunction resulting in a number of disease states (1). Recently, we have learned that many of the cell ...

The biologic activities of protein kinases result from phosphorylation of their substrate proteins. Phosphorylation of proteins causes changes in structure, stability, enzymatic activity, ability to interact with other molecules, or subcellular localization, leading to r ...

Determining in vivo substrates phosphorylated by different mitogen-activated protein kinases (MAPKs) is critical to understanding how these enzymes regulate cell division, differentiation, and growth. The evolution of new technologies, particularly mass spectroscop ...

The mitogen-activated protein kinase (MAPK) signaling pathway is initiated by diverse stimuli that occur at the cell membrane and result in a change in pattern of gene expression in the nucleus. Although multiple proteins are involved in the transmission process through a complex cascade ...

The recent combination of several technology platforms has revolutionized the analysis of expressed cellular proteins (proteomics). Large-scale separation and visualization of proteins is accomplished using two-dimensional gel electrophoresis (2-DE). Proteins are s ...

Deuterium exchange, also called hydrogen exchange, has been used for decades in experiments aimed at elucidating structural information and investigating protein folding (1–4). Deuterium exchange takes place when any acidic proton in a protein molecule exchanges for a deuteron in b ...

Sequential activation of kinases (protein kinase cascades) is a common mechanism of signal transduction in many cellular processes (1). Over the past decade several related intracellular signaling cascades have been elucidated, collectively known as mitogen-activated prot ...