Single Molecule Enzymology Fluorescence Based And High Throughput Methods

Single Molecule Enzymology  Fluorescence Based and High Throughput Methods PDF
Author:
Publisher: Academic Press
ISBN: 0128095474
Size: 56.18 MB
Format: PDF, Mobi
Category : Medical
Languages : un
Pages : 616
View: 4616

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Single-Molecule Enzymology, Part A, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in single-molecule enzymology Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion

Methods In Enzymology

Methods in Enzymology PDF
Author: Maria Spies
Publisher:
ISBN:
Size: 44.60 MB
Format: PDF, ePub, Mobi
Category : Fluorescence spectroscopy
Languages : un
Pages : 618
View: 6486

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Single Molecule Enzymology Nanomechanical Manipulation And Hybrid Methods

Single Molecule Enzymology  Nanomechanical Manipulation and Hybrid Methods PDF
Author:
Publisher: Academic Press
ISBN: 0128095032
Size: 19.43 MB
Format: PDF, Docs
Category : Science
Languages : un
Pages : 484
View: 1362

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Single-Molecule Enzymology, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in single-molecule enzymology Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion

Development Of Molecular Tools And High Throughput Screens In Protein Engineering

Development of Molecular Tools and High Throughput Screens in Protein Engineering    PDF
Author: Kok Hong (Sean) Lim
Publisher:
ISBN:
Size: 62.35 MB
Format: PDF, Kindle
Category :
Languages : un
Pages : 226
View: 2456

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This dissertation represents an effort of to utilize various protein engineering approaches and methods, together with the understanding of the basic of protein biochemistry, to design novel molecular tools and high throughput screening methods for the study and characterization of the interactions between proteins, a protein and a ligand, as well as a protein and a peptide. The stability and activity of streptavidin depend upon the oligomerization of the four identical subunits. Therefore, disruption to subunit association through mutations at the subunit interface compromises both the stability and activity of the protein. To restore the function and stability of protein upon subunit dissociation, we described a systematic strategy for the construction of streptavidin monomers and dimers.^The mutations that were rationally engineered by minimizing binding pocket flexibility and stabilizing originally buried native dimer interface through the introduction of disulfide bond and salt bridges on subunit interface, have resulted in substantial improvement in both binding affinity and thermostability of the protein, compared to previously engineered mutants that lack these stabilizing mutations. These engineered streptavidin monomer and dimer mutants were shown to label biotinylated receptors on the cell surface efficiently, which demonstrates their applicability in the field of molecular detection. To further improve the binding affinity and thermalstability of the protein, we adopted protein homology modeling approach to create a hybrid of two avidin like proteins, streptavidin and rhizavidin. The resulting hybrid, named as mStrav, has greatly improved binding affinity by approximately 44-fold and thermalstability by approximately 28 oC.^We showed that mStrav (1) can be used as a detection tool for recognizing surface immobilized biotinylated ligand, (2) can be expressed stably on yeast surface and mammalian surface to trap biotinylated ligands, and (3) can be fused to fluorescence proteins (EGFP and YPet) to create bifunctional molecules capable of monovalent biotin detection. Such versatile streptavidin monomer, mStrav, should be a useful reagent for designing novel detection systems based on biotin recognition. Obtaining a molecular description of protein-protein interactions (PPI) is important to build an accurate model of structure-function relationship. Detailed models of PPI can also facilitate the development of novel molecular reagents to regulate key biological processes. The formation of protein complexes is critical for carrying out biological functions, hence, it is useful to design a screening platform that can be used to analyze interactions between proteins.^Yeast display is a versatile platform for high throughput protein engineering and was used to study a broad range of proteins, including antibodies, receptors, enzymes, and model proteins. However, displaying unstable and transient protein complexes on yeast surface is challenging, especially for those protein complexes that weakly associate with each other and has a high dissociation constant. We demonstrated that an engineered intersubunit disulfide bond can be used to covalently crosslink transient and unstable protein complexes displayed on the yeast display. The displayed complex can be verified and quantified by flow cytometry. We also demonstrated that the formation of an intersubunit disulfide bond is highly specific and depends on brings the interacting cysteine residues to close proximity.^Together, we showed that disulfide trapping can be used to stabilize transient and unstable protein complexes and the combination with yeast surface and flow cytometry may be useful for studying protein-protein interaction . In the last section of the dissertation, we evaluated specific enzyme-substrate relationship based on measuring the fluorescence resonance energy transfer (FRET) that varies with the level of posttranslational modification (PTM). The use of FRET for the detection of PTM has been demonstrated in previous studies, which showed that the strategy is general and can be used to study a large array of posttranslational modifications on a common platform. However, these methods used in these studies are not applicable to a high throughput analysis of enzyme-substrate relationship because they use low throughput methods, such as fluorescence spectroscopy or microscopy for monitoring the changes in fluorescence.^In this study, we have demonstrated that the substrate specificity of a PTM enzyme can be characterized in vitro and in cell using a genetic FRET detector, which consists of a short peptide linked with a phosphotheorine binding protein, sandwiched by two fluorescence proteins that can be detected and quantified by flow cytometry. This study shows that flow cytometry can be used to quantify FRET efficiency to a near single residue resolution and therefore, is useful in identifying the sequences that are preferentially targeted for PTM. Such high throughput assay can be used as a robust molecular tool to characterize the mechanism of substrate recognition during various biologically relevant PTM processes.

Pharmacogenetics

Pharmacogenetics PDF
Author: Wendell Weber
Publisher: Oxford University Press
ISBN:
Size: 33.39 MB
Format: PDF, Mobi
Category : Medical
Languages : en
Pages : 433
View: 757

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Genes are important modifiers of the human response to drugs, hormones, and toxins. In this second edition of 'Pharmacogenetics', Weber brings together laboratory methods and epidemiologic studies in defining the role of heredity in human drug response.

Fluorescence Methods And Applications

Fluorescence Methods and Applications PDF
Author: Otto S. Wolfbeis
Publisher: Wiley-Blackwell
ISBN:
Size: 39.10 MB
Format: PDF, ePub, Mobi
Category : Science
Languages : en
Pages : 452
View: 3422

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This volume features papers on new spectroscopic methods and techniques, the development and application of fluorescent probes, and new techniques and applications of fluorescence imaging. Specific areas include the following: fluorescence lifetime, fluorescence (in vivo) imaging, time-resolved fluorescence, luminescence anisotropy, fluorescent (NMIR) labels, luminescent lanthanides, fluorescent sensors and probes, fluorescence microscopy, FRET, fluorescent nanoparticles and dots, high-throughput screening, fluorescent bioassays, luminescence-based DNA technologies, FISH and immunohistochemistry, luminescence on metal surfaces, fluorescent proteins, upconversion, multiphoton fluorescence, confocal techniques, near-field and far-field techniques, single photon counting, fluorescence correlation spectroscopy (FCS), and flow cytometry. NOTE: Annals volumes are available for sale as individual books or as a journal. For information on institutional journal subscriptions, please visit www.blackwellpublishing.com/nyas. ACADEMY MEMBERS: Please contact the New York Academy of Sciences directly to place your order (www.nyas.org). Members of the New York Academy of Science receive full-text access to the Annals online and discounts on print volumes. Please visit http://www.nyas.org/MemberCenter/Join.aspx for more information about becoming a member

Expression Genetics

Expression Genetics PDF
Author: Michael McClelland
Publisher: Eaton Publishing Company
ISBN:
Size: 16.20 MB
Format: PDF, Docs
Category : Science
Languages : en
Pages : 379
View: 4071

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Inexpensive handheld device for the construction of high-density nucleic acid arrays. (M. Schummer, W.-I. Ng, P. S. Nelson, R. E. Bumgarner, and L. Hood). Update by M. Schummer. Adapting the Biomek 2000 laboratory automation workstation for printing DNA microarrays. (J. Macas, M. Nouzová, and D. W. Galbraith) Update by J. Macas, M. Nouzová, and D. W. Galbraith. Parallel production of oligonucleotide arrays using membranes and reagent jet printing. (D. I. Stimpson, P. W. Cooley, S. M. Knepper, and D. B. Wallace) Update by D. I. Stimpson. Agarose-embedded tissue arrays for histologic and genetic analysis. (G. S. Tsao-Wu, C. H. Weber, L. R. Budgeon, and K. C. Cheng) Update by K. C. Cheng, L. G. Beckwith and X. Wang. Using oligonucleotide probe arrays to access genetic diversity. (R. J. Lipshutz, D. Morris, M. Chee, E. Hubbell, M. J. Kozal, N. Shah, N. Shen, R. Yang, and S. P. A. Fodor) Update by M. Hurt. Direct hybridization of large-insert genomic clones on high-density gridded cDNA filter arrays. (S. Kern and G. M. Hampton) Update by L. C. Amler, S. Kern, and G. M. Hampton. TurboPrep II: and inexpensive, high-throughput plasmid template preparation protocol. (D. S. Konecki and J. J. Phillips) Update by D. S. Konecki and J. J. Phillips. Amplification of mRNAs from single, fixed, TUNEL-Positive Cells. (D. M. O'Dell, R. Raghupathi, P. B. Crino, B. Morrison 3, J. H. Eberwine, and T. K. MsIntosh) Update by D. M. O'Dell, R. Raghupathi, P. B. Crino, B. Morrison 3, J. H. Eberwine, and T. K. MsIntosh. Laser capture microdissection of single cells from complex tissues. (C. A. Suarez-Quian, S. R. Goldstein, T. Phida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, and R. F. Bonner) B Fingerprinting Methods. Non-Radioisotopic AFLP Protocol Applied to Honey Bee (Apis mellifera L.) DNA. (A. Suazo and H. G. Hall). Chemiluminescent Detection of APLP Markers. (J.-J. Lin, J. Ma, and J. Kuo). DAF Optimization using tagushi methods and the effect of thermal cycling parameters on DNA amplification. (G. Caetano-Anollés). C. subtraction. High-Stringency substraction for the identification of differentially regulated cDNA clones. (C. P. Scutt and P. M. Gilmartin). Differential screening of a subtracted cDNA library: a method to search for genes preferentially expressed in multiple tissues. (H. Jin, X. Cheng, L. Diatchenko, P. D. Siebert, and C.-C. Huang). Update by L. Diatchenko, S. Trelogan, and P. D. Siebert. Isolation of differentially expressed genes by combining representational difference analysis (RDA) and cDNA library arrays. (M. Geng, C. Wallrapp, F. Muller-Pillasch, M. Frohme, J. D. Hoheisel, and T. M. Gress). Update by M. M. Geng, C. Wallrapp, F. Muller-Pillasch, M. Frohme, J. D. Hoheisel, and T. M. Gress. D. cDNA array applications. High-Throughput cDNA screening utilizing a low order neural network filter. (G. M. Huang, J. Farkas, and L. Hood). Update by G. M. Huang. Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes. (R. Ross, X.-L. Ross, B. Rueger, T. Laengin, and A. B. Reske-Kunz). Update by R. Ross, X.-L. Ross, B. Rueger, T. Laengin, and A. B. Reske-Kunz. Representative cDNA libraries and their utility in gene expression profiling. ( W. O. Endege, K. E. Steinmann, L. A. Boardman, S. N. Thibodeau, and R. Schlegel). E. Quantitative RT-PCR. High-Throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure. One-step fluorescent probe product-enhanced reverse transcriptase assay. (B. A. Arnold, R. W. Hepler, and P. M. Keller). Update by B. A. Arnold, R. W. Hepler, and P. M. Keller. Flow Cytometric quantification of surface-displayed recombinant receptors on staphylococci. (C. Andréoni, L. Goetsch, C. libon, P. Samuelson, T. N. Nguyen, A. Robert, M. Uhlén, H. Binz, and S. Stahl). Update by C. Andréoni, L. Goetsch, C. libon, P. Samuelson, T. N. Nguyen, A. Robert, M. Uhlén, H. Binz, and S. Stahl. Gene VIII-Based, Phage-Display vectors for selection against complex mixtures of ligands. (K. Jacobsson and L. Frykberg) Update by K. Jacobsson and L. Frykberg. Biopanning phage display libraries using magnetic beads Vs. Polystyrene Plates. (S. J. McConnell, T. Dinh, M.-H. Le, and D. G. Spinella). Fusion proteins could generale false positives in peptide phage display. (K. K. Murthy, I. Ekiel, S.-H. Shen, and D. Banville). Update by K. K. Murthy, I. Ekiel, S.-H. Shen, and D. Banville. Phosphorylation-Directed antibodies in high-flux screens for compounds that modulate signal transduction. (J. A. Alberta and C. D. Stiles). Update by J. A. Alberta and C. D. Stiles. B. Two-Hybrid selection. Mammalian Two-Hybrid system: A complementary approach to the yeast two-hybrid system. ( Y. Luo, A. Batalao, H. Zhou, and L. Zhu). Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression. (K. J. Fuller, M. A. Morse, J. H. M. White, S. J. Dowell, and M. J. Sims). Update by K. J. Fuller, M. A. Morse, J. H. M. White, S. J. Dowell, and M. J. Sims. Use of a dicistronic expression cassette enconding the green fluorescent protein for the screening and selection of cells expressing inducible gene products. (D. D. Mosser, A. W. Caron, L. Bourget, P. Jocolieur, and B. Massie). Update by D. D. Mosser, A. W. Caron, L. Bourget, P. Jocolieur, and B. Massie. Dual-function reporte protein for analysis of gene expression in living cells. (R.N. Day, M. Kawecki and D. Berry). Fusion of green fluorescent protein with the Zeocin Resistance marker allows visual screening and drug selection of transfected euraryotic cells. (R.P. Bennett, C.A. Cox, J.P. Hoeffler). Update by R.P. Bennett, C.A. Cox, J.P. Hoeffler. Green fluorescent protein tag for studies of drug induced translocation of nuclear protein RH-II/Gu. (B.C. Valdez, L. Perlaky, Z.-J. Cai, D. Henning, and H. Busch). Fission yeast expression vectors adapted for positive identification of gene insertion and green fluorescent protein fusion. (Y. Zhao, R.T. Elder, M. Chen, and J. Cao). Update by Y. Zhao, R.T. Elder, M. Chen, and J. Cao. Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene. (R.R. Muldoon, J. P. Levy, S.R. Kain, P.A. Kitts, and C.J. Link Jr). A combined selection and reporter gene for retroviral and transgenic studies. (J. Blake, P. C. Salinas, and S. M. hughes). Retroviral gene transfer in chondrogenic limb bud micromass cultures. (N. S. Stott, Y.-S. Lee, and C.-M. Chuong). Update by W.-P. Wang, N. Kasahara, and C.-M. Chuong. High-efficiency gene transfer and pharmacologic selection of genetically engineered human keratinocytes. (H. Deng, K.A. Choate, Q. Lin, and P.A. Khavary).

Life Science Automation Fundamentals And Applications

Life Science Automation Fundamentals and Applications PDF
Author: Mingjun Zhang
Publisher: Artech House Publishers
ISBN:
Size: 28.79 MB
Format: PDF
Category : Medical
Languages : en
Pages : 507
View: 3196

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This comprehensive resource provides a solid grounding in life science and automation engineering essentials and describes state-of-the-art techniques for the design and development of sensors and actuators, lab-on-a-chip and bio-MEMs platforms, and more.