细胞功能测定

Phosphoinositides are a vitally important class of intracellular-signaling molecules that regulate cellular processes, including signaling through cell-surface receptors, remodeling of the cytoskeleton, vesicle-mediated protein trafficking, and various nucl ...

Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2α (cPLAα) and for secreted PLA2s (sPL ...

The emerging technique of bioluminescence resonance energy transfer (BRET) allows us to detect protein interactions in live cells and in real time, thus providing a new window into cellular signal transduction processes. We present experimental protocols for expressing fusion pr ...

Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photoble ...

A wide variety of methods used in the study of signal transduction in eukaryotes rely on the ability to precipitate proteins from whole cell extracts. Immunoprecipitation and related methods of affinity purification are routinely used to assess binding partner interactions and enzy ...

Signal transduction pathways mediate cell-cell interactions and integrate signals from the extracellular environment through specific receptors at the cell membrane. They play a pivotal role in regulating cellular growth and differentiation and in mediating many physiolo ...

Translocation of proteins from the mitochondrial intermembrane space to the cytoplasm is a critical event during apoptosis. There are several methods for assaying this event cited in the literature. In this chapter, we highlight separation of cytosolic and mitochondrial fractions ...

In eukaryotic organisms gene expression is regulated through a variety of upstream transacting factors (transcription factors) whose primary function appears to be the targeting of coregulatory protein complexes, which interact with basal transcription machinery to define ...

Two-dimensional electrophoresis (2-DE) is a powerful technique to differentially display patterns of protein expression and posttranslational modifications, providing a good strategy to monitor molecular responses induced by the activation or inactivation of specific ...

Controlling the stability of cellular proteins is a fundamental way by which cells regulate growth, differentiation, survival, and development. Measuring the turnover rate of a protein is often the first step in assessing whether or not the function of a protein is regulated by proteolysis u ...

Protein kinases, encoded by approx 2% of eukaryotic genes, represent one of the major classes of cell-regulatory molecules. Assessment of the catalytic activity of a specific protein kinase can be an important step in elucidating signal-transduction pathways that affect cell behavio ...

Methods are presented for purifying bovine testes calmodulin and the calmodulin-regulated plasma-membrane calcium ATPase from human erythrocytes by calcium dependent affinity chromatography. The assay of CaM Kinase II using a synthetic peptide substrate is also described.

Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. Th ...

Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into phy ...

In the 20 yr since the discovery of proteins whose levels oscillate during the cell cycle in marine invertebrate embryos (1), the study of cyclins and their cognate protein kinases has revealed a wealth of information on how eukaryotic cells control cyclical functions connected with cell prol ...

It is becoming apparent that an increase in the rate of protein degradation represents one mechanism for the regulation of key proteins during the cell cycle (1). The importance of protein degradation in the control of cell proliferation is elegantly illustrated by the regulated synthesis a ...

Immunofluorescence is one of the most widely used techniques to study the localization of transcription factors, proteins, and structural components of nuclear architecture and cytoarchitecture. High-resolution in situ immunofluorescence approaches permit assessme ...

This technique is used to visualize sites of active transcription in a permeabilized cell and does not require radiolabeled molecules (e.g., Uridine). Nonradioactive ribonucleic acid (RNA) precursors (e.g., 5-bromouridine-5′-triphosphate ) are used and can be detected by using fluo ...

The resolution that can be attained in assessment of the intranuclear localization of cellular proteins is dependent on specificity of the antibodies. Primary antibodies should be well characterized. We recommend testing antibody specificity by immunoblotting. In general, mon ...

Only a handful of tagged molecules have been used successfully in vivo to monitor gene expression. The most commonly used are β-galactosidase (β-gal) and green fluorescent protein (GFP). Both molecules pose limitations with in vivo detection. When using GFP, autofluorescence may be encou ...