实验基础

Standardization of bacterial culture is crucial for in vivo experiments addressing Helicobacter/host �interaction. Here we present methods for bacteria culture and infection of mice.

Animal models are essential for in vivo analysis of Helicobacter-related diseases. Transgenic mice and Mongolian gerbil models have been the corner stone of present research focusing on both bacterial virulence factors and host response to infection. Establishing a reproducible ...

Helicobacter pylori CagA and VacA are two critical virulence factors that modulate disease severity in the infected host. The following chapter outlines methods employed to study the effects of these virulence factors on several key signaling pathways in epithelial cells.

Adherence and internalization of Helicobacter pylori into epithelial cells is a recently recognized event in the pathogen’s life cycle.

The precise quantitative determination of the different lipid classes in mutant cells is key to understand the possible role of the respective gene product in lipid homeostasis. In this chapter, we describe methods based on thin-layer chromatography that are employed routinely to dete ...

Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along ...

Two-dimensional gel electrophoresis (2-DE) offers the opportunity of separating several hundred proteins from a total yeast cellular extract. A detailed description is provided here of the different steps required for the separation and visualization of radiolabeled yeast pro ...

Quantitative extraction of lipids from the tissue or microorganism of choice is key to their subsequent analysis. In this chapter, we describe a simple and rapid protocol that relies on glass bead disruption in the presence of organic solvents to quantitatively extract lipids from yeast cel ...

Often preparations of isolated organelles contain other, unwanted, cellular components. For biochemical experiments to determine the localization of newly identified proteins, or to determine the whole set of proteins (or the proteome) from a desired organelle, these unwanted co ...

Peroxisomes are ubiquitous subcellular organelles of eukaryotic cells. As with all organelles, peroxisomes can be purified from cell lysates using a combination of differential centrifugation and density gradient centrifugation. Here, we describe a method for purifying pero ...

Mitochondria fulfill a large variety of metabolic tasks such as respiration, beta-oxidation, heme biosynthesis, ketone-body, or amino acid synthesis. In addition to their metabolic role, mitochondria are also key players in cellular apoptosis and participate in the generation of re ...

The yeast two-hybrid system is a poauwerful molecular genetic tool conceived by Fields and Song (1). The article is a comprehensive set of methods designed to take the reader through a yeast two-hybrid analysis of your favorite gene (YFG). This article details the preparation for a screen, the scre ...

The accurate replication and expression of genetic information is ultimately governed by the interaction of regulatory proteins with specific sites on chromosomes. In recent years, our understanding of how these interactions occur in vivo has advanced considerably, in large part o ...

This chapter aims to provide the reader with a one-stop reference to the basic procedures needed to grow, store, mate, and sporulate yeast cells. Starting with recipes for the different types of media, the chapter then goes on to explain how cells are grown to the appropriate cell numbers at the correct st ...

The mechanisms of biological chromatin assembly and their regulation have been studied intensively using cellular extracts, particularly those from the embryonic cells of various metazoans. Here we describe how to prepare and use a crude chromatographic fraction from budding yea ...

Synthetic lethality occurs when the combination of two mutations leads to an inviable organism. Screens for synthetic lethal genetic interactions have been used extensively to identify genes whose products buffer one another or impinge on the same essential pathway. For the yeast Sacc ...

The neutral/neutral (N/N) two-dimensional (2-D) agarose gel technique is a useful tool for understanding the mechanisms leading to the complete duplication of linear eukaryotic chromosomes. For the yeast Saccharomyces cerevisiae, it has been used to localize and characterize orig ...

Intracellular localization is important for the characterization of a gene product. Microscopy of fluorescent protein fusions has become the method of choice to define the spatial and temporal behavior of a protein. We show here that recombinant antibody fluorescent protein fusions ...

Fluorescence microscopy is the essential technique for investigation of the intracellular distribution of macromolecules and various organelles also in yeast cells. In this chapter, detailed practical procedures for fluorescence microscopic observations developed or ...

The technique for the transformation of Saccharomyces cerevisiae using the LiAc/SS Carrier DNA/PEG method is described. We describe a rapid method, for use when large numbers of transformants are not necessary. A high-efficiency method for the generation of large numbers of transforma ...