生物化学技术

Potassium channels play an essential role in a wide range of biological processes, including cell volume regulation and the maintenance and control of electrical signals. With the advent of the structural era of ion channel biology, it has become critical to learn more about the functional pr ...

KCNQ1 is a voltage-activated potassium channel α -subunit expressed in various cell types, including cardiac myocytes and epithelial cells. KCNQ1 associates with different β-subunits of the KCNE protein family. In the human heart, KCNQ1 associates with KCNE1 to generate the I Ks current c ...

Radioimmunoassay (RIA) and enzyme immunoassay (EIA) are used for detecting very small amounts of a hormone, drug, or other such material in blood (serum), tissue, urine, or culture media. Both radioimmunoassay and enzyme immunoassay techniques for the measurement of eicosanoids are bas ...

The sequencing of the genomes of many different species has greatly helped our understanding of organelles. This information has driven the development of mass spectrometry (MS)-based methods for large-scale analysis of proteins. One of these methods uses two-dimensional liquid ch ...

Chromosomes are supermolecules that contain most of the DNA within a cell and are visible under optical and electron microscopes. Although they were observed at the earliest stage of genetics, their fundamental structure is not yet understood. The reasons for this are debated among resear ...

All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytop ...

This chapter presents procedures for preparation and analysis of fractions enriched in plasma membranes from frozen tissue. It consists of a method for extraction and fractionation of membranes and a method for enzymatic digestion of membrane proteins without use of detergents. The me ...

Integral membrane proteins are represented by 20–30% of the eukaryotic genome and crucial for cellular functions including cell signaling, nutrient influx, toxin efflux, and maintenance of osmotic balance. Importantly, over 70% of all drugs are targeted at membrane proteins. Because ...

Peroxisomes take part in various metabolic pathways related to the regulation of lipid homeostasis. Although detailed information on the enzymes involved in the peroxisomal lipid metabolism was acquired in the past, the mechanisms of metabolic exchange between peroxisomes and the ...

Gas-phase fractionation (GPF), defined as iterative mass spectrometric interrogations of a sample over multiple smaller mass-to-charge (m/z) ranges, enables the ions selected for collision-induced dissociation to come from a greater number of unique peptides compared to the ions ...

Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping m ...

This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which a ...

Synaptic vesicles store and subsequently release neurotransmitters into the synaptic cleft thereby regulating chemical neurotransmission in the brain. Proteins present in synaptic vesicles vary greatly in structure and function and have been identified primarily by gene ...

Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades arranged in a pinwheel-shaped array. Centrioles are required for recruitment of pericentriolar material (PCM) during centrosome formation, and they act as basal bodies, which ...

Pancreatic zymogen granules (ZGs) are specialized for digestive enzyme storage and regulated secretion in exocrine pancreas and are a classical model for studying secretory granule function. To understand the function of this organelle, we have conducted a proteomic study to ident ...

The 20S proteasome is a multicatalytic protein complex present in all eukaryotic cells. Associated to regulatory complexes, it plays a major role in cellular protein degradation and in the generation of Major Histocompatibility Complex (MHC) class I antigenic peptides. In mammalian c ...

This chapter describes the purification of ribosomal particles from a mutant strain of Escherichia coli using sucrose gradients and the characterization of their protein composition by a combination of mass spectrometry (MS) techniques. The main objective is to identify the riboso ...

Sub-cellular proteomics has proven to be a powerful approach to link the information contained in sequenced genomes from eukaryotic cells to the functional knowledge provided by studies of cell compartments. Chloroplasts are plant-specific organelles and are the site of photosyn ...

Although a great deal is known about the structure and function of most mammalian organelles, comprehensive proteomes are necessary to provide a molecular framework to integrate this information. The Golgi complex is the central organelle of the secretory pathway and functions to post ...

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to ...