细胞功能测定

Blue native electrophoresis (BNE) is a long established method for the analysis of native protein complexes. Applications of BNE range from investigating subunit composition, stoichiometry, and assembly of single protein complexes to profiling of whole complexomes. BNE is an indi ...

The planar lipid bilayer technique is a powerful experimental approach for electrical single channel recordings of pore-forming membrane proteins in a chemically well-defined and easily modifiable environment. Here we provide a general survey of the basic materials and procedu ...

The isolation and functional reconstitution of large membrane protein complexes is an important step towards the biochemical characterization of such sophisticated molecular machines. Reconstitution is a multistep process that requires the mild solubilization of membr ...

The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to ...

In vitro import experiments with isolated organelles are a powerful tool for investigation of the biogenesis of proteins. A key issue in such experiments is an assay to distinguish between correctly and incorrectly imported proteins. Here we describe an assay to monitor in vitro the proper me ...

The development of small-interfering RNA (siRNA)-mediated gene-silencing strategies has made it possible to study the transport of precursors of soluble and membrane proteins into the endoplasmic reticulum (ER) of human cells. In these approaches, a certain target gene is silenced in ...

With the availability of increasing numbers of fluorescent protein variants and state-of-the-art imaging techniques, live cell microscopy has become a standard procedure in modern cell biology. Fluorescent markers are used to visualize the dynamic processes that take place in liv ...

The ability to bind to and translocate across lipid bilayers is of paramount importance for the extracellular administration of intracellularly active compounds in cell biology, medicinal chemistry, and drug development. A combination of the so-called uptake and release experim ...

This chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound d ...

Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-l-phenylalanine (pBpa), a photorea ...

Fluorescence correlation spectroscopy (FCS) is an emerging technique employed in biophysical studies that exploits the temporal autocorrelation of fluorescence intensity fluctuations measured in a tiny volume (in the order of fL). The autocorrelation curve derived from the f ...

Natural and synthetic membrane active peptides as well as fragments from membrane proteins interact with membranes. In several cases, such interactions cause the insertion of the peptides to the membrane and their assembly within the lipid bilayer. Here we present spectroscopic appr ...

The interaction between membrane proteins and their (protein) ligands is conventionally investigated by nonequilibrium methods such as co-sedimentation or pull-down assays. Surface Plasmon Resonance can be used to monitor such binding events in real-time using isolated memb ...

The interaction of proteins with biological membranes is a key factor in their biogenesis and proper function. Hence, unraveling the properties of this interaction is very important and constitutes an essential step in deciphering the structural and functional characteristics of a m ...

The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the non ...

The topology of integral membrane proteins with a weak topological tendency can be influenced when fused to tags, such as these used for topological determination or protein purification. Here, we describe a technique for topology determination of an untagged membrane protein. This tec ...

The complexity of biological membranes presents technical challenges for the analysis of membrane protein biogenesis and function. Here we describe an in vitro fluorescence-based experimental approach for studying the high-resolution structural features of membrane pro ...

The crystallization of membrane proteins is an essential technique for the determination of atomic models of three-dimensional structures by X-ray crystallography. The compositions of solutions of purified membrane proteins are altered, so as to transiently induce supersatu ...

Electron crystallography is a powerful technique for studying the structure and function of membrane proteins, not only in the ground state, but also in active conformations. When combined with high-resolution structures obtained by X-ray crystallography, electron crystallog ...

The yeast Saccharomyces cerevisiae has become a valuable eukaryotic model organism to study biochemical and cellular processes at a molecular basis. A common strategy for such studies is the use of single and multiple mutants constructed by genetic manipulation which are compromised ...