Live-cell fluorescent biosensors for activated signaling proteins

A new generation of live-cell fluorescent biosensors enables us to go beyond visualization of protein movements, to quantify the dynamics of many different protein activities. Alternate approaches can report post-translational modifications, ligand interactions and conformational changes, revealing how the location and subtle timing of protein activity controls cell behavior.     

Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression

This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides (or whole antigens) and monensin during a 6- to 24-h stimulation period. (Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.     

A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.     

Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6–8 M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4 M urea. The activity of rGST was assayed in 2 M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.     

Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP.

In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed.     

Protein chip based miniaturized assay for the simultaneous quantitative monitoring of cancer biomarkers in tissue extracts

A multiplexed fluorescence immunoassay using a novel planar waveguide technology-based microarray system, ZeptoMARK (Zeptosens), was developed to detect simultaneously urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues. The three analytes assay was cross-validated with single-analyte ELISA/chemiluminescence immunosorbent assay tests, revealing good correlations and enhanced assay sensitivities (LODs) of 1 pg/mL for uPA, 33 pg/mL for PAI-1, and 1 pg/mL for VEGF. Values were well within the 80-120% limits for assay recovery and within the +/-20% limits for assay precision. The uPA, PAI-1, and VEGF results obtained from 50 breast cancer cytosols using the protein array system demonstrated that the microarray-based multiplexed assay is a sensitive and robust tool to be used for the simultaneous quantification of cancer markers in small breast cancer tissue samples (core biopsies). The miniaturized, multiplexed assay format has a potential to be used for the quantitative analysis of a larger set of validated markers with significance in disease management.     

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Of RNA-binding proteins and their targets: interaction determines expression

Combining the prediction of interactions between mRNAs and RNA-binding proteins with experimental expression profiles uncovers novel regulatory paradigms concerning proliferation and differentiation processes.See related research, http://genomebiology.com/2014/15/1/R13     

根据基因表达谱筛选间接互作蛋白质进行蛋白质功能预测

利用基因表达谱数据,通过计算互作蛋白质的表达相关系数,来筛选、优化蛋白质互作网络。结果显示,利用经过筛选的互作数据,根据邻居计数法和卡方法进行功能预测的预测效果明显提高,距离待测蛋白质较远的邻居也包含着与待测蛋白质功能一致的信息。     

A sensitive assay for proteins and biliproteins

An assay for proteins in dilute solution is described which is based on binding of Coomassie blue G-250 to proteins. The dye-protein complex formed is separated out from solution by centrifugation. The absorbance of the redissolved precipitate in 70:30 ($\text{v}\text{v}$) Tris:methanol is monitored at 605 nm. The assay is standardized for biliprotein determination.     

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.     

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of γ-secretase are discussed.     

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.