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Backbone Dynamics of Free Barnase and Its Complex with Barstar Determined by 15N NMR Relaxation Study
Journal of Biomolecular NMR, 18: 107–118, 2000. KLUWER/ESCOM 107 © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study Sarata C. Sahua, Abani K. Bhuyanb, Jayant B. Udgaonkarb & R.V. Hosura;∗ aDepartment of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India; bNational Centre for Biological Sciences, Tata Institute of Fundamental Research, University of Agricultural Sciences (UAS) - Gandhi Krishi Vigyan Kendra (GKVK) Campus, Bangalore 560 065, India Received 18 April 2000; Accepted 7 July 2000 Key words: backbone dynamics, barnase, barstar, complex, 15N NMR relaxation, protein–protein interactions Abstract Backbone dynamics of uniformly 15N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 ◦C, pH 6.6, using 15N relaxation data obtained from proton-detected 2D {1H}-15N NMR spec- 15 troscopy. N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear {1H}-15N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N- terminal residue) backbone amide 15N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (τm) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. -
Blotting Techniques Blotting Is the Technique in Which Nucleic Acids Or
Blotting techniques Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. Blotting techniques are used to identify unique proteins and nucleic acid sequences. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research. General principle The blotting methods are fairly simple and usually consist of four separate steps: electrophoretic separation of protein or of nucleic acid fragments in the sample; transfer to and immobilization on paper support; binding of analytical probe to target molecule on paper; and visualization of bound probe. Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography. -
Characterization of in Vitro Oxidized Barstar
CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector FEBS 15911 FEBS Letters 370 (1995) 273-277 Characterization of in vitro oxidized barstar C. Frisch, G. Schreiber, A.R. Fersht* Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, UK Received 13 July 1995 mutant which has been solved independently by Guillet et al. Abstract The polypeptide inhibitor of the ribonuclease barnase, [8] to 2.6 A resolution and by Buckle et al. [4] to 2.0/k resolu- barstar, has two cysteine residues in positions 40 and 82. These tion. The distance between the s-carbon atoms of the alanines have been proposed to form a disulfide bridge leading to an in position 40 and 82 in the two crystal structures of the barstar increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and (C40A/C82A) double mutant is 11.4 A [8] and 11.7 A [4], re- the biochemical and physico-chemical properties of the oxidized spectively. In the solution structure of barstar that was deter- monomers were analysed. The oxidized proteins show no inhibi- mined by NMR [7] (Fig. 1), the distance between the s-carbon tion of barnase using a plate assay and are significantly destabi- atoms of the cysteines is 11.5 h. These s-carbon distances are lized. CD spectra indicate a loss of secondary structure. The outside the normal range for a disulfide bond, which is usually amino acid substitution E80 --~ A stabilizes the oxidized barstar from 4.4 to 6.8 A [9]. -
Southern Blotting Teacher’S Guidebook
PR139 G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected] A Geno Technology, Inc. (USA) brand name Southern Blotting Teacher’s Guidebook (Cat. # BE-315) think proteins! think G-Biosciences www.GBiosciences.com MATERIALS INCLUDED ....................................................................................................... 3 SPECIAL HANDLING INSTRUCTIONS ................................................................................... 3 ADDITIONAL EQUIPMENT REQUIRED ................................................................................ 3 TIME REQUIRED ................................................................................................................. 3 AIMS .................................................................................................................................. 4 BACKGROUND ................................................................................................................... 4 TEACHER’S PRE EXPERIMENT SET UP ................................................................................ 5 PREPARATION OF AGAROSE GEL ................................................................................... 5 PREPARE THE DNA LADDER ........................................................................................... 5 PREPARE DILUTE DNA STAINING SOLUTION .................................................................. 5 MATERIALS FOR DEMONSTRATION ................................................................................... 6 DEMONSTRATION -
Bioanalytical Chemistry 4. Gel Electrophoresis
73 Bioanalytical chemistry 4. Gel Electrophoresis Required reading: Sections 9.1, 9.2.3, 9.2.4, 9.5.1, 10.1 to 10.7, 11.1 to 11.5, and 15.5 of Mikkelsen and Cortón, Bioanalytical Chemistry Some objectives for this section ⇒ You will know what DNA agarose gel electrophoresis is ⇒ You will know what the difference between normal and pulsed field electrophoresis of DNA ⇒ You will know what SDS-PAGE is. ⇒ You will understand how the basis for molecular basis for size-based separation of proteins by SDS- PAGE. ⇒ You will know what IEF is. ⇒ You will know how SDS-PAGE and IEF can be combined in 2-dimensional gel electroophoresis ⇒ You will know what a Western Blot is. ⇒ You will appreciate how these techniques can be used in the analysis of DNA and proteins. Primary Source Material • Chapter 4 and 6 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (NCBI bookshelf). • Chapter 3 and 7 of Molecular Cell Biology 4th ed. (Ch. 9, 5th ed.): Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (NCBI bookshelf). • Chapter 12 of Introduction to Genetic Analysis Anthony: J.F. Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart (NCBI bookshelf). • Some animations are from http://www.wiley-vch.de/books/info/3-527-30300-6/. • http://www.piercenet.com/ Electrophoresis 74 The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f). -
Blotting Techniques M.W
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector RESEARCH TECHNIQUES MADE SIMPLE North, South, or East? Blotting Techniques M.W. Nicholas1 and Kelly Nelson2 Journal of Investigative Dermatology (2013) 133, e10; doi:10.1038/jid.2013.216 One of the cornerstones of modern molecular biology, blotting is a powerful and sensitive technique for identifying WHAT BLOTTING DOES the presence of specific biomolecules within a sample. • Blotting allows specific and sensitive detection of a Subtypes of blotting are differentiated by the target protein (western) or specific DNA or RNA sequence molecule that is being sought. The first of these tech (Southern, northern) within a large sample isolate. niques developed was the Southern blot, named for Dr. • Targets are first separated by size/charge via gel Edwin Southern, who developed it to detect specific DNA electrophoresis and then identified using a very sequences (Southern, 1975). Subsequently, the method was sensitive probe. modified to detect other targets. The nomenclature of these • Variations of these techniques can detect post techniques was built around Dr. Southern’s name, resulting translational modifications and DNAbound proteins. in the terms northern blot (for detection of RNA), western blot (for detection of protein), eastern blot (for detection • Western blotting may also be used to detect a of posttranslationally modified proteins), and south circulating antibody in a patient sample or confirm western blot (for detection of DNA binding proteins). Most an antibody’s specificity. researchers consider the eastern blot and the southwestern blot variations of western blots rather than distinct entities. -
The Folding of Groel-Bound Barnase As a Model for Chaperonin-Mediated Protein Folding (Chaperone/Protein Engineering)
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5326-5330, June 1995 Biochemistry The folding of GroEL-bound barnase as a model for chaperonin-mediated protein folding (chaperone/protein engineering) FERNANDO J. CORRALES AND ALAN R. FERSHT* Medical Research Council Unit for Protein Function and Design and Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 lEW, United Kingdom Contributed by Alan R. Fersht, March 3, 1995 ABSTRACT We have analyzed the pathway of folding of solution, then the rate of folding should fall to zero when all barnase bound to GroEL to resolve the controversy ofwhether the barnase becomes bound at saturating concentrations of proteins can fold while bound to chaperonins (GroEL or GroEL. Cpn6O) or fold only after their release into solution. Four We now analyze the folding pathway in detail by stopped- phases in the folding were detected by rapid-reaction kinetic flow monitoring the change of tryptophan fluorescence of measurements of the intrinsic fluorescence of both wild type barnase, GroEL not having tryptophan residues. Unfortu- and barnase mutants. The phases were assigned from their nately, the fluorescence of denatured barnase when bound to rate laws, sensitivity to mutations, and correspondence to GroEL is so close to that of fully folded barnase that it is regain ofcatalytic activity. At high ratios ofdenatured barnase difficult to monitor the folding by using conventional stopped- to GroEL, 4 mol ofbarnase rapidly bind per 14-mer of GroEL. flow fluorimeters. We have now overcome these difficulties by At high ratios of GroEL to barnase, 1 mol of barnase binds replacing Trp-94 by tyrosine (mutant W94Y), which removes with a rate constant of 3.5 x 107 s-1 M-1. -
Investigating the Structural Determinants of Electrostatic Binding Among Protein-Protein Complexes: a Systematic, Large-Scale Computational Study
Investigating the Structural Determinants of Electrostatic Binding among Protein-protein Complexes: A Systematic, Large-scale Computational Study Emma Nechamkin April 2012 Wellesley College Wellesley, MA Submitted in Partial Fulfillment of the Prerequisites for Honors in Chemistry Acknowledgements Writing my senior thesis has been a wonderful experience, and there are many people to whom I owe thanks for support and guidance. First and foremost, my brilliant advisor Mala Radhakrishnan has been incredibly supportive and understanding. In dorkier terms, her advising style is incredibly close to the hypothetical optimum. Without her constant guidance, motivation, and feed- back, my thesis simply would not exist. I am truly grateful to be a student in her lab. Second, the faculty on my thesis committee, David Haines and Don Elmore of the Chemistry Department, and YuJin Ko of the English Department, have dedicated significant time to my thesis. In particular, the chemistry faculty on my committee have provided insight on much of my work and offered much needed encouragement. Third, I would like to thank the entire Wellesley College Chemistry Department. Clearly the best department on campus, the chemistry department has an unparal- leled involvement with and dedication to its students. At a sometimes stressful and overwhelming school like Wellesley, I am glad to have found my niche. Fourth, each member of the Radhakrishnan Lab over my time here has taught me something about research and listened to at least one incredibly long story I have shared. For the group's helpfulness, reassurance, and companionship, I am deeply grateful. In particular, Ying Yi Zhang has shared some wonderful code with me. -
Non-Radioactive Southern Blot Reagents Store As Labeled
Product Specifications Non-Radioactive Southern Blot Analysis, Electrophoresis Reagents Polymerase Chain Reaction Reagents, Omni-Pure Reagents, Hybridization and Detection Reagents, Custom Primers and Probes Non-Radioactive Southern Blot Reagents Store as labeled. For research use only. Catalog Number Description Unit Size 40-3010-10 Agarose LE Molecular Biology Grade 100 gms 40-5023-20 Lumisol II Hybridization Solution; for non-toxic hybridizations; 200 mL 200 mL 40-5024-20 Lumisol III Hybridization Solution; for oligonucleotide probes; 200 mL 200 mL 40-5010-10 CDP-Star® Substrate; Ready-to-Use 0.25 mM in spray bottle; 10 mL 10 mL 40-5020-25 Hybwash A, Hybridization Wash Solution Concentrate( 20X SSC); 250 mL 250 mL 40-5021-10 Hybwash B, Hybridization Wash Solution Concentrate (10% SDS); 100 mL 100 mL 40-5025-20 Maleic acid buffer 10X (Buffer M 10X); 200 mL 200 mL 40-5025-50 Maleic acid buffer 10X (Buffer M 10X); 500 mL 500 mL 40-5026-10 Blocking solution for hybridization (10%); 100 mL 100 mL 40-5031-10 AP Detection buffer 10X (Alkaline Phosphatase buffer); 100 mL 100 mL 40-5034-10 Depurination Solution (2X) for Southern Blotting; 1 L 1 L 40-5035-10 Denaturation Solution (2X) for Southern Blotting; 1L 1 L 40-5036-10 Neutralization Solution (2X) for Southern Blotting; 1L 1 L Lumisol II Hybridization Solution; for non-toxic hybridizations; 200 mL Catalog Number 40-5023-20 Lumisol III Hybridization Solution; for oligonucleotide probes; 200 mL Catalog Number 40-5024-20 Storage: Room Temperature. Below 22°C precipitates may appear. Warm to 40°C and gently swirl to dissolve the precipitates. -
FOR Environmental Release of Genetically Engineered Mustard
Assessment of Food and Environmental Safety of GE mustard ASSESSMENT OF FOOD AND ENVIRONMENTAL SAFETY (AFES) FOR Environmental release of Genetically Engineered Mustard (Brassica juncea) hybrid DMH-11 and use of parental events (Varuna bn3.6 and EH2 modbs2.99) for development of new generation hybrids Application submitted by Centre for Genetic Manipulation of Crop Plants (CGMCP), University of Delhi South Campus, New Delhi 2016 1 Assessment of Food and Environmental Safety of GE mustard 2 Assessment of Food and Environmental Safety of GE mustard Table of Contents 1 CHAPTER 1: INTRODUCTION 5 1.1 The Application 6 1.2 Global status of hybrid seed production technology in Brassica napus using MS-RF system deploying the genes used for B. juncea 8 2 CHAPTER 2: BIOLOGY OF INDIAN MUSTARD 11 2.1 Origin and domestication of Brassica juncea 11 2.2 Brassica species present in India and their distribution 12 2.3 Cultivation of B. juncea: soil and climate requirement 14 2.4 Standard agriculture practices for growing B. juncea 14 2.5 Weeds, Major pest and disease 15 2.6 Zonalization of varietal testing 15 3 CHAPTER 3: INDIAN BIOSAFETY REGULATORY FRAMEWORK 17 3.1 Introduction 17 3.2 Step by step process to be followed by the applicant 22 3.3 Step by step regulatory compliance and data generation in the case of GE mustard parental lines and hybrid DMH-11 24 3.4 Assessment of Food/feed and Environmental Safety (AFES) - Risk Assessment Process 25 4 CHAPTER 4: MOLECULAR CHARACTERIZATION OF GE MUSTARD HYBRID DMH-11 AND ITS PARENTAL LINES 33 4.1 Introduction 33 4.2 The male sterility- fertility restorer technology 34 4.3 Gene constructs 35 4.4 Method of genetic transformation of B. -
A General Approach for the Generation of Orthogonal Trnas
Chemistry & Biology 8 (2001) 883^890 www.elsevier.com/locate/chembiol Research Paper A general approach for the generation of orthogonal tRNAs Lei Wang a; 1, Peter G. Schultz b; * aDepartment of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA bDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Received 26 March 2001; accepted 29 May 2001 First published online 27 July 2001 Abstract Background: The addition of new amino acids to the genetic cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four code of Escherichia coli requires an orthogonal suppressor tRNA mutant suppressor tRNAs were selected that are poorer substrates Tyr that is uniquely acylated with a desired unnatural amino acid by for E. coli synthetases than M. jannaschii tRNACUA, but still can Tyr an orthogonal aminoacyl-tRNA synthetase. A tRNACUA^tyrosyl- be charged efficiently by M. jannaschii TyrRS. Tyr tRNA synthetase pair imported from Methanococcus jannaschii Conclusions: The mutant suppressor tRNACUA together with can be used to generate such a pair. In vivo selections have been the M. jannaschii TyrRS is an excellent orthogonal tRNA^ developed for selecting mutant suppressor tRNAs with enhanced synthetase pair for the in vivo incorporation of unnatural amino orthogonality, which can be used to site-specifically incorporate acids into proteins. This general approach may be expanded to unnatural amino acids into proteins in E. coli. generate additional orthogonal tRNA^synthetase pairs as well as Results: A library of amber suppressor tRNAs derived from M. probe the interactions between tRNAs and their cognate Tyr Tyr jannaschii tRNA was generated. -
Southern and Western Blot Techniques, Dna Profiling
RASAQ NURUDEEN OLAJIDE LECTURE CONTENT INTRODUCTION SOUTHERN BLOTTING WESTERN BLOTTING DNA PROFILING (DNA FINGERPRINTING) PARENTAL TESTING PROCEDURES INTRODUCTION Then the Lord said to Cain, ”Where is your brother Abel?” He said,” I do not know; am I my brother’s keeper?” And the Lord said “What have you done? Listen; your brother’s blood is crying out to me from the ground!”-Genesis 4:9-10 DNA: a true marker of identity FORENSIC SCIENCE Definition – Relating to the use of science or technology in the investigation and establishment of facts or evidence in a court of law. Source – From the Latin word Forum. The Forum was a public gathering place during Roman times, where judicial activities and public business was conducted. HISTORY OF FORENSIC SCIENCE Many people believe that Arthur Conan Doyle was the first to popularize forensic science with his Sherlock Holmes novels. The first, A Study in Scarlet, was published in 1887. WHAT IS BLOTTING? Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN. TYPES OF BLOTTING TECHNIQUES Blotting technique Western blot Southern Blot Northern Blot It is used to detect It is used to detect DNA. It is used to detect RNA. protein. SOUTHERN BLOTTING Professor Sir Edwin Southern, Professor of Biochemistry and Fellow of Trinity developed this method in 1975. Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago.