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Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or -macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca(2+)-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using biotinylated ALG-2. Dependency of Ca(2+) for interaction is examined by inclusion of CaCl(2) or EGTA in buffers used for binding assays.

Cystic fibrosis (CF) is caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as an anion channel and is known to interact with a number of other cellular proteins involved in ion transport. To date more than 1,800 mutations are known, most of which result in various degrees of impaired transport function of the gene product. Due to the high inter-individual variability of disease onset and progression, CF still is a diagnostic challenge. Implemented almost 20 years ago, the measurement of electrolyte transport function of rectal biopsies is a useful ex vivo tool to diagnose CF. In this chapter we will review the different approaches to perform ion transport measurements and try to highlight the advantages and limitations of these techniques.

Proteomics attempts to characterise the gene products expressed in a cell or tissue via a range of biophysical techniques including crystallography and NMR and, more relevantly to this volume, chromatography and mass spectrometry. It is becoming increasingly clear that the native states of segments of many of the cellular proteins are not stable, folded structures, and much of the proteome is in an unfolded, disordered state. These proteins and their disordered segments have functionally interesting properties and provide novel challenges for the biophysical techniques that are used to study them. This chapter focuses on computational approaches to predicting such regions and analyzing the functions linked to them, and has implications for protein scientists who wish to study such properties as molecular recognition and post-translational modifications. We also discuss resources where the results of predictions have been collated, making them publicly available to the wider biological community.

Enhanced expression of transglutaminases is a frequent, though not obligatory phenomenon in apoptosis, which is associated with cells dying in steady interaction with their tissue environment. Modification of cellular proteins by transglutamination is a tightly controlled procedure. If transglutamination is dysregulated, it has profound and potentially detrimental consequences on cellular functioning. Under conditions normally occurring in living cells transglutaminase activity is usually undetectably low (latent) and can only be tested by careful preselection of proteins of interest. In late stages of apoptosis, transglutaminases can become rampant in dying cells and a minuscule fraction of dead cells may overshadow many more living ones, which may cause inherent and severe methodological and interpretation bias. Therefore, in this chapter, we describe the analysis of dead cell remnants for protein-bound transglutaminase-mediated cross-link content. In the techniques described below, we rely on the increasing availability and user-friendliness of mass spectrometric equipments.

Quantitation of specific proteins in complex mixtures is simplified by the use of antibodies directed against the protein of interest. If the specific protein is differentially expressed within a population of cells, quantitation of the protein in cell lysates by immunoblotting will provide an average quantity of the protein per cell. As a result, when lysates of different cell populations are compared, large changes in the amount of a protein in a small percentage of cells cannot be distinguished from small changes in the amount of a protein in a large percentage of cells. Flow cytometric analysis solves this problem by providing a means to measure the amount of a specific protein within each individual cell in a population. A fluorescently labeled probe, usually an antibody, is required for detection of the protein. A single cell suspension is reacted with an antibody and passed through a flow cytometer, which focuses the cell suspension into a stream that intersects a laser beam. As each cell passes through the beam, the fluorescent probe in each cell is excited and photomultiplier tubes register the degree of fluorescence (1). Multivariate analysis, the simultaneous measurement of multiple parameters, is a powerful feature of flow cytometry. When determining the quantity of a specific protein in a cell, the quantity of other molecules in the same cell can also be measured by using additional fluors specifically targeted to other molecules. Examples of second molecules measured simultaneously include DNA (2–4) or additional proteins (5). In addition, measurement of light scatter can provide information about cell size and granularity. Most flow cytometers are capable of measuring six colors plus light scatter, but specialized flow cytometers that are capable of measuring as many as eighteen colors are now available.

Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded. The proteolysis of longlived proteins by macroautophagy is a standard, specific measure of autophagic degradation and represents an end-point assay for the pathway. The assay is based on a pulse-chase approach, whereby cellular proteins are radiolabeled by an isotopically marked amino acid, the short-lived, rapidly turned over, proteins are allowed to be degraded during a long chase period, and then the remaining, stable radiolabeled proteins are subjected to autophagic degradation. The classical application of this method has been in hepatocytes, but the recent growth of interest in autophagy has necessitated adaptation of this method in nonliver cells. Here we describe a protocol to quantify autophagic degradation of longlived proteins in macrophages. This chapter details the method of analyzing autophagic proteolysis in RAW264.7 mouse macrophages.

Methods for generating recombinant vaccinia viruses for the expression of foreign viral glycoproteins in mammalian cell lines and the purification of expressed viral glycoproteins are described. These methods are based on many years of experience with the influenza hemagglutinin glycoprotein (HA). However, they are applicable for studies on other viral glycoproteins, and with slight modifications, could be useful for cellular proteins as well.

Protein phosphatases reverse the covalent modifications of numerous cellular proteins imposed by the activation of protein kinases. Although protein phosphatases generally demonstrate broader substrate specificity than protein kinases, at least in vitro, these enzymes have evolved complex mechanisms to target specific physiological substrates and respond to physiological stimuli to control numerous physiological events. This chapter provides a brief overview of the challenges that faced researchers in protein phosphatases in years past and highlights numerous state-of-the-art techniques (described in greater detail in other chapters in this volume) available to today's scientists. These methods should equip investigators with a rich toolbox of reagents and techniques and promise a brighter future for the study of eukaryotic protein phosphatases. These experimental approaches should also facilitate future investigations directed at unraveling the role of phosphatases in signaling networks in human health and disease.

Investigating the cell biology of gene expression requires methodologies for localizing RNA relative to proteins involved in RNA transcription, processing, and export. Adenovirus is an important model system for the analysis of eukaryotic gene expression and is also being used to investigate the organization of gene expression within the nucleus. Here are described the combined in situ hybridization and immunofluorescence staining techniques that have been used to study the localization of viral RNA relative to nuclear structures that contain splicing factors.

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 under specific physiological conditions. Over the years, procedures to determine the half-life of proteins in cultured eukaryotic cells have been well-established. This chapter describes in detail the two most frequently used methods, pulse-chase analysis and cycloheximide blocking, to determine a protein's half-life in yeast and cultured mammalian cells.

Cellular proteins that interact with p53 play a major role in both positive and negative regulation of this tumor suppressor and can fine-tune its response to specific cellular stresses. As a consequence, p53 biology will not be complete until these interacting proteins are fully characterized. This chapter outlines two methods for identifying and characterizing p53-binding proteins: (i) glutathione-S-transferase (GST) protein-protein interaction assay; and (ii) co-immunoprecipitation (co-IP) assay. These two methods are ideal for any laboratory to perform; the assays are short in duration, do not require specialized expertise to establish in the laboratory, give reliable and reproducible data, and are cost-efficient, because few reagents are needed. This chapter gives a basic description of these two techniques and provides tips that are not found in other protocol manuals on how to achieve the best results.

Large-format 2D gel electrophoresis systems in routine operation are capable of resolving several thousand cellular proteins in 1 or 2 d (1,2). For the last decade, a combination of Edman microsequence analysis and identification of proteins by staining with specific antibodies has been used to systematically identify proteins and establish cellular databases (3–5). There are, however, significant problems associated with these approaches. Most proteins are only present in the low- to upper-femtomole range, which is significantly below the level at which automated sequencers can reliably operate (6,7). The relatively slow speed of the Edman process also means that the number of proteins is too great to permit large-scale characterization within any useful period of time. The use of monoclonal antibodies, while both rapid and sensitive, requires the ready availability of a large pool of specific antibody probes.

Large-format 2D gel electrophoresis systems have been developed that are capable of resolving several thousand cellular proteins in a matter of days [1,2]. For a number of years, a combination of Edman microsequence analysis and identification of proteins by staining with specific antibodies has been used to systematically categorize proteins and establish cellular databases [3, 4, 5]. There are, however, significant problems associated with these approaches. Most resolved proteins are only present in the low- to upper-femtomole range, significantly below the level at which automated sequencers can reliably operate [6, 7]. The relatively slow speed of the Edman process (one or two samples per machine per day) also means that the sheer number of proteins is too great to permit large-scale characterization within any useful period of time. The use of monoclonal antibodies, whilst both rapid and extremely sensitive, requires the ready availability of a large pool of antibody probes.

We have previously shown by gel-shift assayori-p-DNA column chromatography and DNasel foatprinting, 2 species of cellular anti-EBNA1 proteins were produced by BJAB Dells upon activation by TPA (1, 2). These anti-E1 proteins could inhibit, compete and uncouple the sped fic binding of E iA-1 to the oir-p .Under denaturing conditions by SDS-PAGE, the molecular weight of the anti-EBNA1 proteins were 40,000 and 60,000, respectively. Here, we stowed that non-denaturing glycerol-gradient sedimentation was able to isolate the 40 kDa and 60 kDa anti-EBNAl that could bind to the 30 by repeat sequence of the EBV ori-p. Additionally, these anti-EBNA1 proteins were constitutively expressed by epithelial HeLa cells. Competitive gel-shift assay determined that the anti-EBNA1 proteins were not related to c-myc proteins. Our observations suggest that anti-EBNA1 may play a role in the preference for lytic infection when EBV infects epithelial cells, although the generality of anti-EBNAl expression in epithelial cells awaits confirmation.

The EBNA-1 protein has an unusual structure in which more than 30% consists of a repeating glycine-alanine sequence (1). This sequence may also be present in normal cellular proteins (2, 3). The glycine-alanine region may therefore be the basis for the development of autoantibodies during EBV infection. We have investigated the antibodies generated during infectious mononucleosis (IM). We find that IgM autoantibodies occur in all patients and that these antibodies recognize a series of at least 8 normal cellular proteins.