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Labeling Approach (labeling + approach)
Selected AbstractsIn Vivo Imaging of a Bacterial Cell Division Protein Using a Protease-Assisted Small-Molecule Labeling ApproachCHEMBIOCHEM, Issue 5 2008Souvik Chattopadhaya Announce on entry: We present a method for the site-specific labeling of target proteins using a set of cell permeable small-molecule probes. The tobacco etch virus (TEV) NIa protease, was used to generate target proteins with an N-terminal cysteine residue, which was subsequently labeled with thioester probe(s) in a site-specific and covalent manner. Furthermore, we demonstrate the utility of this approach for the study of FtsZ, an important bacterial cell-division protein (see figure). [source] Myopodia (postsynaptic filopodia) participate in synaptic target recognitionDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2003Sarah Ritzenthaler Abstract Synaptic partner cells recognize one another by utilizing a variety of molecular cues. Prior to neuromuscular synapse formation, Drosophila embryonic muscles extend dynamic actin-based filopodia called "myopodia." In wild-type animals, myopodia are initially extended randomly from the muscle surface but become gradually restricted to the site of motoneuron innervation, a spatial redistribution we call "clustering." Previous experiments with prospero mutant embryos demonstrated that myopodia clustering does not occur in the absence of motoneuron outgrowth into the muscle field. However, whether myopodia clustering is due to a general signal from passing axons or is a result of the specific interactions between synaptic partners remained to be investigated. Here, we have examined the relationship of myopodia to the specific events of synaptic target recognition, the stable adhesion of synaptic partners. We manipulated the embryonic expression of ,PS2 integrin and Toll, molecules known to affect synaptic development, to specifically alter synaptic targeting on identified muscles. Then, we used a vital single-cell labeling approach to visualize the behavior of myopodia in these animals. We demonstrate a strong positive correlation between myopodia activity and synaptic target recognition. The frequency of myopodia clustering is lowered in cases where synaptic targeting is disrupted. Myopodia clustering seems to result from the adherence of a subset of myopodia to the innervating growth cone while the rest are eliminated. The data suggest that postsynaptic cells play a dynamic role in the process of synaptic target recognition. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 31,40, 2003 [source] A novel approach to tag and identify geranylgeranylated proteinsELECTROPHORESIS, Issue 20 2009Lai N. Chan Abstract A recently developed proteomic strategy, the "GG-azide"-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the "click" chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The "GG-azide"-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications. [source] Magnetosonoporation: Instant magnetic labeling of stem cellsMAGNETIC RESONANCE IN MEDICINE, Issue 6 2010Bensheng Qiu Abstract The purpose of this study was to develop an instant MR cell labeling technique, called magnetosonoporation. First, a magnetosonoporation apparatus was successfully established for MR labeling of stem cells. Then, the safety of this new cell labeling approach was confirmed by evaluation of cell viability, proliferation, and differentiation of magnetosonoporation-labeled and unlabeled C17.2 neural stem cells. Subsequently, the feasibility of using in vivo MRI to detect magnetosonoporation/Feridex-labeled stem cells was validated in living animals and confirmed by histologic correlation. The magnetosonoporation technique is expected to be convenient, efficient, and safe for future clinical application of MRI-guided cell therapies. Magn Reson Med 63:1437,1441, 2010. © 2010 Wiley-Liss, Inc. [source] Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approachPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2009Peter van Ulsen Abstract Isobaric labeling reagents such as Tandem Mass Tags (TMT®) enable the genome-wide quantification of protein expression levels under different conditions using a gel-free MS/MS-based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron. Expression of 35 proteins was found to be induced or repressed under iron-limiting conditions, including 11 proteins whose ORFs were not previously identified in DNA array studies as being regulated by iron availability at the transcriptional level. These 11 proteins include proteins likely involved in iron metabolism. [source] Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membraneGLIA, Issue 12 2009J. W. Ou Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [source] Improving the Procedure for Detection of Intrahepatic Transplanted Islets by Magnetic Resonance ImagingAMERICAN JOURNAL OF TRANSPLANTATION, Issue 10 2009M. L. Malosio Islet transplantation is an effective therapy for restoring normoglycemia in type-1 diabetes, but long-term islet graft function is achieved only in a minority of cases. Noninvasive magnetic resonance imaging of pancreatic islets is an attractive option for "real-time" monitoring of graft evolution. So far, previous studies have been performed in the absence of a standardized labeling procedure and, besides a feasibility study in patients, the effectiveness and safety of various labeling approaches were tested only with high field magnets (4.7 T). In this study, we addressed: (a) standardization of a labeling procedure for human islets with clinically-approved contrast agent Endorem®, (b) safety aspects of labeling related to inflammation and (c) quality of imaging both at 7 T and 1.5 T. We have highlighted that the ratio of Endorem®/islet is crucial for reproducible labeling, with a ratio of 2.24 ug/IEQ, allowing successful in vivo imaging both with 1.5 T and 7.0 T magnets up to 143 days after intrahepatic transplant. With this standardized labeling procedure, labeled islets are neither inflamed nor more susceptible to inflammatory insults than unlabeled ones. This report represents an important contribution towards the development of a standardized and safe clinical protocol for the noninvasive imaging of transplanted islets in humans. [source] SERS Microscopy: Nanoparticle Probes and Biomedical ApplicationsCHEMPHYSCHEM, Issue 9-10 2009Sebastian Schlücker Prof. Abstract Microspectroscopic imaging: Surface-enhanced Raman scattering (SERS) microscopy is a novel method of vibrational microspectroscopic imaging for the selective detection of biomolecules in targeted research. This review summarizes current designs of nanoparticle-based SERS probes (see figure) and highlights first biomedical applications of SERS microscopy for protein localization ex and in vivo. Surface-enhanced Raman scattering (SERS) microscopy is a novel method of vibrational microspectroscopic imaging for the selective detection of biomolecules in targeted research. This technique combines the advantages of biofunctionalized metal nanoparticles and Raman microspectroscopy for visualizing and quantifying the distribution of target molecules such as proteins in cells and tissues. Advantages of SERS over existing labeling approaches include the tremendous multiplexing capacity, quantification using the characteristic SERS signatures and high photostability. This review summarizes current designs of nanoparticle-based SERS probes and highlights first biomedical applications of SERS microscopy for protein localization ex and in vivo. [source] | ||||||||||||||||