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Methods in Molecular Biology

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https://www.readbyqxmd.com/read/28092054/sh2-domain-histochemistry
#1
Sophia Buhs, Peter Nollau
Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092053/sh2-domain-based-fret-biosensor-for-measuring-bcr-abl-activity-in-living-cml-cells
#2
Mari Fujioka, Yumi Asano, Shigeyuki Nakada, Yusuke Ohba
Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092052/real-time-single-molecule-visualization-of-sh2-domain-membrane-recruitment-in-growth-factor-stimulated-cells
#3
Dongmyung Oh
In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092051/sh2-binding-site-protection-assay-a-method-for-identification-of-sh2-domain-interaction-partners-by-exploiting-sh2-mediated-phosphosite-protection
#4
Joshua A Jadwin
Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092050/microwestern-arrays-for-systems-level-analysis-of-sh2-domain-containing-proteins
#5
Mark F Ciaccio, Richard B Jones
The Microwestern Array (MWA) method combines the scalability and miniaturization afforded by the Reverse Phase Lysate Array (RPLA) approach with the electrophoretic separation characteristic of the Western blot. This technology emulates the creation of an array of small Western blots on a single sheet of nitrocellulose allowing for the sensitive and quantitative measurement of hundreds of proteins from hundreds of cell lysates with minimal cost and maximal accuracy, precision, and reproducibility. The MWA is a versatile technology that can be easily configured for purposes such as antibody screening, cell signaling network inference, protein modification/phenotype regression analysis, and genomic/proteomic relationships...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092049/rosette-assay-highly-customizable-dot-blot-for-sh2-domain-screening
#6
Khong Y Ng, Kazuya Machida
With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092048/using-reciprocal-protein-peptide-array-screening-to-unravel-protein-interaction-networks
#7
Huadong Liu, Courtney Voss, Shawn S C Li
Protein-protein interactions (PPIs) play a central role in almost all cellular processes. Recent technological advances have enabled the elucidation of an incredibly complex PPI network within the cell. However, protein interactions driven by posttranslational modifications (PTMs) such as phosphorylation, which comprises a significant part of the PPI network, have proven difficult to decipher systematically. Herein, we describe a reciprocal protein-peptide array strategy to uncover PPIs mediated by tyrosine phosphorylation and the Src homology 2 (SH2) domain...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092047/analysis-of-the-global-changes-in-sh2-binding-properties-using-mass-spectrometry-supported-by-quantitative-stable-isotope-labeling-by-amino-acids-in-cell-culture-silac-technique
#8
Radoslaw M Sobota
Quantitative mass spectrometry (MS)-based proteomics enables fast and reliable analysis of protein complexes. Its robustness and sensitivity effectively substitute traditional antibody-based approaches. Here, we describe the combination of mass spectrometry and Stable Isotope Labeling by Amino acids in Cell culture (SILAC) in characterization of the SH2 domain binding capacity.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092046/identification-of-tyrosine-phosphorylated-proteins-by-sh2-domain-affinity-purification-and-mass-spectrometry
#9
Sophia Buhs, Helwe Gerull, Peter Nollau
Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092045/sh2-domains-as-affinity-reagents-for-phosphotyrosine-protein-enrichment-and-proteomic-analysis
#10
Mi Ke, Bizhu Chu, Lin Lin, Ruijun Tian
Dynamic tyrosine phosphorylation is a key molecular modulation for many signal transduction events. Because of their low abundance and dynamic nature in cells, the detection and enrichment of phosphotyrosine proteins has long relied on specific antibodies, such as 4G10 and P-Tyr-100. Another well-established approach for phosphotyrosine proteins recognition and enrichment is by their specific binding domains, such as Src homology 2 (SH2) domains. In this chapter, we describe a typical analytical approach for purifying specific SH2 domains, enriching specific phosphotyrosine proteins from activated cells, mass spectrometry analysis, and related data analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092044/high-throughput-quantification-of-sh2-domain-phosphopeptide-interactions-with-cellulose-peptide-conjugate-microarrays
#11
Brett W Engelmann
The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092043/characterizing-sh2-domain-specificity-and-network-interactions-using-spot-peptide-arrays
#12
Bernard A Liu
Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092042/alpha-based-multiplexed-assay-for-identifying-sh2-domain-antagonists
#13
Akira Asai, Kazuyuki Takakuma
Constitutive activation of STAT3/5b frequently occurs in various human malignancies. STAT3/5b activation involves dimerization via intermolecular pTyr-SH2 binding; therefore, antagonizing this interaction is a feasible approach to inhibit STAT3/5b activation for cancer therapy. We have developed a multiplexed assay to assess STAT3- and STAT5b-SH2 binding in a single well by combining AlphaLISA and AlphaScreen beads. In this chapter, we describe application of the method for the purpose of identifying new STAT3 and STAT5b antagonists...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092041/in-solution-sh2-domain-binding-assay-based-on-proximity-ligation
#14
Kazuya Machida
Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092040/binding-assays-using-recombinant-sh2-domains-far-western-pull-down-and-fluorescence-polarization
#15
Kazuya Machida, Bernard Liu
Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092039/calorimetric-measurement-of-sh2-domain-ligand-affinities
#16
Marissa A McKercher, Deborah S Wuttke
Isothermal titration calorimetry (ITC) has emerged as a leading approach in the characterization of protein/ligand interactions. This technique measures the heat change of a system upon binding of a ligand to a biomolecule, and thereby requires no immobilization, intrinsic fluorescence, or labeling of any kind of either species. If properly designed, a single experiment can not only measure the binding affinity, but also determine additional binding and thermodynamic parameters, including the enthalpy, entropy, and the stoichiometry of the interaction...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092038/nmr-chemical-shift-mapping-of-sh2-peptide-interactions
#17
Marissa A McKercher, Deborah S Wuttke
Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments offer a rapid and high resolution approach to gaining binding and conformational insights into a protein-peptide interaction. By tracking (1)H and (15)N chemical shift changes over the course of a peptide titration into isotopically labeled protein, amide NH pairs of amino acids whose chemical environment changes upon peptide binding can be identified. When mapped onto a structure of the protein, this approach can identify the peptide-binding interface or regions undergoing conformation changes within a protein upon ligand binding...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092037/structural-characterization-of-monomeric-dimeric-state-of-p59-fyn-sh2-domain
#18
Radu Huculeci, Fabien Kieken, Abel Garcia-Pino, Lieven Buts, Nico van Nuland, Tom Lenaerts
Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59(fyn) SH2 domain.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092036/creation-of-phosphotyrosine-superbinders-by-directed-evolution-of-an-sh2-domain
#19
Haiming Huang, Tomonori Kaneko, Sachdev S Sidhu, Shawn S C Li
Commercial antibodies raised against phosphotyrosine have been widely used as reagents to detect or isolate tyrosine-phosphorylated proteins from cellular samples. However, these antibodies are costly and are not amenable to in-house production in an academic lab setting. In this chapter, we describe a method to generate super-high affinity SH2 domains, dubbed the phosphotyrosine superbinders, by evolving a natural SH2 domain using the phage display technology. The superbinders are stable and can be easily produced in Escherichia coli in large quantities...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28092035/functionally-altered-sh2-domains-for-biochemical-studies-loss-of-function-mutant-and-domain-concatenation
#20
Mari Ogiue-Ikeda, Kazuya Machida
Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies...
2017: Methods in Molecular Biology
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