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Current Protocols in Cell Biology

Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
March 3, 2017: Current Protocols in Cell Biology
Yanyang Tang, Ji Qiu, Matthias Machner, Joshua LaBaer
We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST...
March 3, 2017: Current Protocols in Cell Biology
Ivan T Rebustini
The increasing repertoire of microRNAs expressed during organ development and their role in regulating organ morphogenesis provide a compelling need to develop methods to assess microRNA function using various in vitro and in vivo experimental models. Methods to assess microRNA function during organ morphogenesis include transfection of microRNA inhibitors (antagomirs) and activators (mimics) into mouse embryonic explanted organs using liposomes, which can potentially result in low efficiency of transfection and off-target effects...
March 3, 2017: Current Protocols in Cell Biology
Xin-Hua Liao, Alan R Kimmel
Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence-based phenotypic assay in a 1536-well plate format for high-throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds...
March 3, 2017: Current Protocols in Cell Biology
Ioannis M Roussis, Fiona A Myers, Garry P Scarlett
Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer...
March 3, 2017: Current Protocols in Cell Biology
Valerie Le Sage, Alessandro Cinti, Andrew J Mouland
Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins...
December 1, 2016: Current Protocols in Cell Biology
Hui Wang, Klaus M Hahn
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered...
December 1, 2016: Current Protocols in Cell Biology
Dawn L Brasaemle, Nathan E Wolins
Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc.
September 1, 2016: Current Protocols in Cell Biology
Laura L Listenberger, Andrea M Studer, Deborah A Brown, Nathan E Wolins
Excess lipid is stored in intracellular organelles known as lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets in fixed or live cells with BODIPY 493/503 are included. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology...
June 1, 2016: Current Protocols in Cell Biology
Janusz Franco-Barraza, Dorothy A Beacham, Michael D Amatangelo, Edna Cukierman
Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface...
June 1, 2016: Current Protocols in Cell Biology
Jiyun Kim, Kandice Tanner
This protocol describes a way to introduce topography to three-dimensional (3D) biomaterials. The self-assembling behavior of magnetic particles can be exploited to form nanoscale to microscale fibers, such that one can dissect the contribution of topography on cell behavior, which is independent of other physical properties of the biomaterial (e.g., stiffness). The magnetic particles are chemically cross-linked with several extracellular matrix (ECM) proteins and then using magnetic force-mediated assembly, one can program aligned nanofibers in a 3D hydrogel...
March 1, 2016: Current Protocols in Cell Biology
Michael Timaner, Ofrat Beyar-Katz, Yuval Shaked
The tumor microenvironment consists of a variety of cell types. The contribution of each cell type to the tumor is an emerging subject in the field of cancer research. Here, we describe protocols for dissociating tumor tissues and Matrigel plugs into single cells for further analysis by flow cytometry. These protocols can be used for evaluating the cellular component of solid tumors from human or mouse origin or Matrigel plugs implanted in mice. The protocols describe the dissociation of tumor tissue with or without dissociation automatic devices...
March 1, 2016: Current Protocols in Cell Biology
Vira V Artym
The stroma of invasive tumors becomes enriched in dense fibrillar collagen as a result of the desmoplastic reaction. This desmoplastic collagen exerts profound effects on tumor and normal cells. In view of these findings, it is important to develop novel in vitro cell systems that mimic this desmoplastic extracellular matrix in order to permit cell studies under in vivo-like conditions. This unit provides a protocol and troubleshooting guide for preparation of high-density fibrillar collagen (HDFC) matrices that closely model the desmoplastic collagenous matrix of malignant tumors...
March 1, 2016: Current Protocols in Cell Biology
Mutsuki Amano, Tomoki Nishioka, Yoshimitsu Yura, Kozo Kaibuchi
Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology...
2016: Current Protocols in Cell Biology
Andrew D Doyle
Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel-bundled long fibrils by changing collagen polymerization temperature...
2016: Current Protocols in Cell Biology
Tzu-Chi Chen, Chi-Ying F Huang
Understanding signaling pathway networks via protein-protein interactions (PPIs) at the cellular level is a significant task that has not yet been completed. Here, a systems approach that computationally infers interlinked pathways from numerous PPIs is described. The endogenous PPIs can be empirically detected using an in situ proximity ligation assay (PLA), which detects and visualizes endogenous PPIs and post-translational modifications of proteins with a high sensitivity and specificity. This unit includes two parts: (1) conversion of gene lists into PPIs for investigation and (2) large-scale detection and analysis of endogenous PPIs for elucidating pathway networks...
2016: Current Protocols in Cell Biology
Juan S Bonifacino, David C Gershlick, Esteban C Dell'Angelica
Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein-protein interactions. Clear step-by-step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody. © 2016 by John Wiley & Sons, Inc...
2016: Current Protocols in Cell Biology
Julie G Donaldson
This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope...
December 1, 2015: Current Protocols in Cell Biology
John M Graham
This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients...
December 1, 2015: Current Protocols in Cell Biology
Marvin Bentley, Gary Banker
Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking...
December 1, 2015: Current Protocols in Cell Biology
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