Springer protocols feed by Protein Science 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.LC-MALDI-TOF/TOF for Shotgun Proteomics
Automated liquid chromatography tandem mass spectrometry (LC-MS/MS) is a well-established technique for identification of components from complex mixtures in shotgun proteomics experiments. Approaches involving the use of matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI-MS/MS) comprise the preparation of protein extracts, their enzymatic digestion, the separation of the resulting peptides by nanoscale liquid chromatography coupled to a collector that deposits the microfractions onto a MALDI plate, and finally the mass spectrometry analysis of the fractions. Using an LC-MALDI strategy, the rate of the...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Top-Down Proteomics by Means of Orbitrap Mass Spectrometry
Top-down proteomics has become a popular approach for the analysis of intact proteins. The term “top down” has been coined for the analysis of proteins not involving any enzymatic or chemical cleavage but rather the ionization of the protein as a sound molecule and mass analysis of intact species and fragment ions thereof produced upon dissociation inside a mass spectrometer. One or several charge states of the protein are mass-isolated and subjected to dissociation (MS/MS) in the gas phase. The obtained fragment masses, predominantly from cleavages of the protein along its amino acid backbone, are directly rel...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Analysis of Protein Structure by Cross-Linking Combined with Mass Spectrometry
Cross-linking combined with mass spectrometry is a powerful technique to study protein structure. Here, we present an optimized protocol for the preparation, processing, and analysis of a protein sample cross-linked with isotopically coded, affinity-enrichable, and CID-cleavable cross-linker CyanurBiotinDimercaptoPropionylSuccinimide using LC/ESI-MS/MS on a Thermo Scientific Orbitrap mass spectrometer. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Survey of Shotgun Proteomics
Proteins provide the verbs to biology, and proteomics provides the nouns for their analytical and discovery-driven studies. The term proteomics was coined in the 1990s and deals with the protein complement of the genome—the proteome. Following the classical proteomics era, the development of new mass spectrometric methods for peptide analysis permitted the identification of proteins in peptide mixtures obtained by proteolytic digestion of complex samples, e.g., shotgun proteomics. Since its introduction, shotgun proteomics became the standard technique for the analysis of protein hydrolyzates in a high-throughput way...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
A Roadmap to Applying Optogenetics in Neuroscience
Optogenetics allows for the specific manipulation of the activity of genetically defined cell populations in the CNS. Yet, it requires effective gene delivery, light stimulation, and readout strategies. Here, we provide a roadmap aimed at guiding the experimenter in the process of establishing an optogenetic approach tailored to a given research hypothesis in the field of neuroscience. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Manipulation of Plasma Membrane Phosphoinositides Using Photoinduced Protein–Protein Interactions
We describe the development of these tools using the dephosphorylation of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) as an example and show how they can be used to rapidly and reversibly deplete the plasma membrane of this lipid. We also provide instructions for image analysis. The CRY2–CIB1 dimerization method has also already been adapted for the acute and spatially restricted generation of PI(3,4,5)P3 in the plasma membrane. More generally, this methodology should be broadly applicable to studies of the spatiotemporal regulation of membrane lipid metabolism in many types of cells. (Source: S...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins
The coupling of light-inducible protein–protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to t...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photoconversion of CFP to Study Neuronal Tissue with Electron Microscopy
Being able to use versatile light microscopy on live or fixed samples followed by electron microscopy imaging for high resolution analyses is a challenging goal. The advantage is of course that tracing and localizing fluorescently labeled molecules yields great information about dynamic cellular processes, while electron microscopy of the same sample provides exquisite information about subcellular structures. Here, I describe the straightforward combination of both methods by photoconversion of diaminobenzidine (DAB) through cyan fluorescent protein (CFP) tagged proteins localized to the Golgi apparatus in primary hippoca...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photocontrol of AMPA Receptors with a Photochromic Ligand
Photochromic ligands (PCLs), recently introduced by our group as a tool for researchers in neuroscience, offer the ability to control native receptors with light in a reversible fashion without the need for any genetic manipulation. Here we describe the application of the PCL Azo-Tetrazole-AMPA-3 (ATA-3) to reversibly gate native AMPA-receptors with blue light and thereby control the activity of cortical neurons in brain slices. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photoswitching of Cell Surface Receptors Using Tethered Ligands
We describe here the design of light-gated ionotropic and metabotropic glutamate receptors, the selection of a site for cysteine placement, the method for PTL attachment, and a detailed protocol of photoswitching experiments in cultured cells. These descriptions can guide applications of Chemical Optogenetics to other receptors and serve as a starting point for use in more complex preparations. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Optochemical Activation of Kinase Function in Live Cells
Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases in conju...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Modification of Purified Proteins with Photochemical Protection Compounds for High-Resolution Photoactivation of Protein Function In Vitro and In Vivo
Specific and targeted photoactivation of protein function inside cells, tissues, or whole organisms can be achieved with reversible inhibition of proteins by conjugation with photolabile protection compounds (“caging”). In vitro caging of proteins is thought to cause sterical or functional hindrance of amino acid side chains that are important for protein activity. Following the modification, the caged protein is introduced into the biological system and high-resolution irradiation ensures specific release of protein function in the desired areas. Here, I describe the entire caging procedure and highlight a few...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photoinduced Damage Resulting from Fluorescence Imaging of Live Cells
The widespread application of fluorescence microscopy to study live cells has led to a greater understanding of numerous biological processes. Many techniques have been developed to uniquely label structures and track metabolic pathways using fluorophores in live cells. However, the photochemistry of nonnative compounds and the deposition of energy into the cell during imaging can result in unexpected and unwanted side effects. Herein, we examine potential live cell damage by first discussing common imaging considerations and modalities in fluorescence microscopy. We then consider several mechanisms by which various photoc...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
pcSOFI as a Smart Label-Based Superresolution Microscopy Technique
Stochastic optical fluctuation imaging (SOFI) is a superresolution imaging technique that uses the flickering of fluorescent labels to generate a microscopic image with a resolution better than what the diffraction limit allows. Its adaptation towards fluorescent protein-labeled samples (called photoconversion SOFI or pcSOFI) allows for a straightforward and easily accessible way of generating superresolution images. In this protocol, we will discuss how so-called “smart labels,” and specifically the reversibly switchable fluorescent proteins, have opened doors towards superresolution imaging in general and we ...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photoactivatable Fluorescent Proteins for Super-resolution Microscopy
Super-resolution fluorescence microscopy techniques such as simulated emission depletion (STED) microscopy and photoactivated localization microscopy (PALM) allow substructures, organelles or even proteins within a cell to be imaged with a resolution far below the diffraction limit of ~200 nm. The development of advanced fluorescent proteins, especially photoactivatable fluorescent proteins of the GFP family, has greatly contributed to the successful application of these techniques to live-cell imaging. Here, we will illustrate how two fluorescent proteins with different photoactivation mechanisms can be utilized in high r...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
