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Biochemistry and the Genomic Revolution 1.1
Dedication About the authors Preface Tools and Techniques Clinical Applications Molecular Evolution Supplements Supporting Biochemistry, Fifth Edition Acknowledgments I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. DNA Illustrates the Relation between Form and Function 1.2. Biochemical Unity Underlies Biological Diversity 1.3. Chemical Bonds in Biochemistry 1.4. Biochemistry and Human Biology Appendix: Depicting Molecular Structures 2. Biochemical Evolution 2.1. Key Organic Molecules Are Used by Living Systems 2.2. Evolution Requires Reproduction, Variation, and Selective Pressure 2.3. Energy Transformations Are Necessary to Sustain Living Systems 2.4. Cells Can Respond to Changes in Their Environments Summary Problems Selected Readings 3. Protein Structure and Function 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains 3.3. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops 3.4. Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar Cores 3.5. Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures 3.6. The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure Summary Appendix: Acid-Base Concepts Problems Selected Readings 4. Exploring Proteins 4.1. The Purification of Proteins Is an Essential First Step in Understanding Their Function 4.2. Amino Acid Sequences Can Be Determined by Automated Edman Degradation 4.3. Immunology Provides Important Techniques with Which to Investigate Proteins 4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods 4.5. -
SOP Template Northern Blotting with P-32
Standard Operating Procedure Procedure Northern Blotting using 32P Department Location SOP Prepared By: Section 1: Purpose Northern blotting is a standard method for the detection and quantification of RNA from a cell. This is done by isolating and purifying RNA and using a radioactively-labeled DNA or RNA probe to hybridize to and detect the RNA. Using this technique, temporal and spatial location of RNA expression can be found8. Unlike RT-PCR (real-time PCR), which quantifies the amount of RNA or DNA at various times, Northern blotting can be used not only to quantify but also determine the size of the RNA. It is also useful to perform Northern blotting when studying transfer RNA (tRNA), which appears as the two lowest bands on a gel1. The probes used in Northern blotting do not have to be radioactive, but radioactive probes still have the greatest sensitivity. Quantifying mRNA of tRNA can provide insight into gene expression in cells that are exposed to different environments. Section 2: Personal Protective Equipment and Survey Equipment PPE: • Nitrile Gloves • Lab coat or lab gown • Proper enclosed shoes • Safety glasses Other Equipment: • Geiger counter • Personal chest dosimeter Section 3: Radioactive Material 32P, specific type varies according to what procedure is followed for probe design Supplier: Perkin Elmer Starting activity: 10 mCi/mL Typical use quantities: no more than 10uCi per experiment • Radioactive material (RAM) benchtop exposure time: ~10-20 minutes Activity used per experiment: _______________ RAM handling time: _______________ Frequency of experiment: _______________ Section 4: Potential Hazards • 32P is a high-energy beta emitter and has a half-life of 14.29 days. -
Glycoprotein Detection with the Odyssey Infrared Imaging System
Glycoprotein Detection with the Odyssey ® Infrared Imaging System Julie A. Champoux, Kristi L.H. Ambroz, Joseph B. Hwang, William M. Volcheck, and Amy R. Schutz-Geshwender LI-COR Biosciences, Lincoln, NE 68504 INTRODUCTION bination of lectins can also be used to dissect the Glycosylation is one of the most common and carbohydrate composition of a target protein. important events in post-translational modifica- For more information about IRDye products tion, with over half of all proteins believed to be used in this study, go to http://www.licor.com/bio/ glycosylated.1 Cellular glycoconjugates play reagents/irdyes.jsp. important roles in many biological processes and have been implicated in cancer development, MATERIALS AND METHODS retrovirus infection and other diseases. Carbohy- Sensitivity of biotinylated Con A / IRDye drate-binding proteins known as lectins bind to 800CW-streptavidin and IRDye 800CW-Con A specific oligosaccharides, and can serve as markers Two-fold serial dilutions of α2-macroglobulin, to identify certain cell types or cellular compo- glucose oxidase and RNAse B (Sigma Cat# nents. Lectins have very high binding specificity M6159, 49178 and NEB Cat# P7817S were and have been used to characterize and purify mixed and separated on 4-12% polyacrylamide oligosaccharides.2 This application note describes Tris-glycine gels (Novex/Invitrogen). Samples the use of lectins to detect glycoproteins in West- were electrophoretically transferred onto nitro- ern blot format using the Odyssey® Infrared cellulose membrane (Osmonics). For all blots in Imaging System. this study, blocking was carried out with Odyssey Concanavalin A (Con A) is a carbohydrate- Blocking Buffer (LI-COR, Part # 927-40000) for 1 h binding protein that binds specifically to the at room temperature; washing was performed in most commonly occurring sugars: α-D-mannose, PBST (PBS + 0.1% Tween®-20) for 4 x 5 min, fol- α-D-glucose and, with lower affinity, α-N-acetyl- lowed by a 5 min rinse in PBS before imaging. -
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. -
Near-Infrared Fluorescent Northern Blot
Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Near-infrared fluorescent northern blot BRET R. MILLER,1,4 TIANQI WEI,1,4 CHRISTOPHER J. FIELDS,1,2 PEIKE SHENG,1,2 and MINGYI XIE1,2,3 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA 2UF Health Cancer Center, University of Florida, Gainesville, Florida 32610, USA 3UF Genetics Institute, University of Florida, Gainesville, Florida 32610, USA ABSTRACT Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present al- ternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of dif- ferent RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis. -
NORTHERN BLOT SEM.I Dr.Ramesh Pathak the Northern Blot Is Also
NORTHERN BLOT SEM.I Dr.Ramesh Pathak The Northern blot is also called RNA blot. It is a technique used in molecular biology to study gene expression by detection of RNA in a sample. The northern blot technique developed by James Alwine, David Kemp and Jeorge Stark at Stanford University with contribution from Gerhard Heinrich in 1977.Northern blotting takes its name from its similarity to the first blotting technique, the southern blotting named after biologist Edwin Southern. Procedure The blotting process starts with extraction of total RNA from a homogenized tissue sample or cells on agarose gel containing formaldehyde which is a denaturing agent for the RNA to limit secondary structure. Eukaryotic mRNA can then be isolated through the use of oligo(dT) cellulose chromatography to isolate only those RNAs with a poly A tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter matrix, the RNA samples, now separated by size, are transferred to nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge, is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contain formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperature, which could cause RNA degradation. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. -
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 -
Gene Expression Detection Methods RNA
Gene expression detection methods RNA Dr. Ingrid Müller - AG von Laer 1 TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION Four different human cells - same genes, different structures and functions due to differential gene expression 2 THE CENTRAL DOGMA • Flow of Information: DNA Replication Transcription Translation A B Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through proteins synthesis. When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein. Levels in gene activation may vary between cancerogenic and healthy cells. 3 MOLECULAR METHODS TOOL BOX I. Analysis II. Overexpression III. Inhibition •Western Blot •(adding proteins to in •pharmacological inhibitors, •Proteomics vitro reactions) •dominant-negative Protein •immuno- proteins, histochemisty • protein depletion using •protein chimera antibodies •Electrophoresis •microinjection •RNAi & •Northern Blot •siRNA •RNAse protection •Morpholinos RNA assays •Microarrays •RT-PCR •RNA in-situ •Reporter genes •(microinjection) •Knock-out •Electroporation, •Integrational mutagenesis DNA (gene) •lipofection, •Classic genetics •transgenics 4 RNA METHODS: ELECTROPHORESIS 5 PURIFICATION OF MESSENGER RNA USING OLIGO DT COLUMNS Total cellular RNA; apply at room temperature to Break open anneal polyA tail to oligo(dT) cells in the presence of RNAse Oligo(dT) inhibitors attached to AAAA. -
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). -
Clinical Molecular Genetics in the Uk C.1975–C.2000
CLINICAL MOLECULAR GENETICS IN THE UK c.1975–c.2000 The transcript of a Witness Seminar held by the History of Modern Biomedicine Research Group, Queen Mary, University of London, on 5 February 2013 Edited by E M Jones and E M Tansey Volume 48 2014 ©The Trustee of the Wellcome Trust, London, 2014 First published by Queen Mary, University of London, 2014 The History of Modern Biomedicine Research Group is funded by the Wellcome Trust, which is a registered charity, no. 210183. ISBN 978 0 90223 888 6 All volumes are freely available online at www.history.qmul.ac.uk/research/modbiomed/ wellcome_witnesses/ Please cite as: Jones E M, Tansey E M. (eds) (2014) Clinical Molecular Genetics in the UK c.1975–c.2000. Wellcome Witnesses to Contemporary Medicine, vol. 48. London: Queen Mary, University of London. CONTENTS What is a Witness Seminar? v Acknowledgements E M Tansey and E M Jones vii Illustrations and credits ix Abbreviations xi Ancillary guides xiii Introduction Professor Bob Williamson xv Transcript Edited by E M Jones and E M Tansey 1 Appendix 1 Photograph, with key, of delegates attending The Molecular Biology of Thalassaemia conference in Kolimbari, Crete, 1978 88 Appendix 2 Extracts from the University of Leiden postgraduate course Restriction Fragment Length Polymorphisms and Human Genetics, 1982 91 Appendix 3 Archival material of the Clinical Molecular Genetics Society 95 Biographical notes 101 References 113 Index 131 Witness Seminars: Meetings and Publications 143 WHAT IS A WITNESS SEMINAR? The Witness Seminar is a specialized form of oral history, where several individuals associated with a particular set of circumstances or events are invited to meet together to discuss, debate, and agree or disagree about their memories. -
Journal of Biomedical Discovery and Collaboration
View metadata, citation and similar papers at core.ac.uk brought to you by CORE Journal of Biomedical Discovery and provided by PubMed Central Collaboration BioMed Central Case Study Open Access The emergence and diffusion of DNA microarray technology Tim Lenoir*† and Eric Giannella† Address: Jenkins Collaboratory for New Technologies in Society, Duke University, John Hope Franklin Center, 2204 Erwin Road, Durham, North Carolina 27708-0402, USA Email: Tim Lenoir* - [email protected]; Eric Giannella - [email protected] * Corresponding author †Equal contributors Published: 22 August 2006 Received: 09 August 2006 Accepted: 22 August 2006 Journal of Biomedical Discovery and Collaboration 2006, 1:11 doi:10.1186/1747-5333-1-11 This article is available from: http://www.j-biomed-discovery.com/content/1/1/11 © 2006 Lenoir and Giannella; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. -
Protein Blotting Guide
Electrophoresis and Blotting Protein Blotting Guide BEGIN Protein Blotting Guide Theory and Products Part 1 Theory and Products 5 Chapter 5 Detection and Imaging 29 Total Protein Detection 31 Transfer Buffer Formulations 58 5 Chapter 1 Overview of Protein Blotting Anionic Dyes 31 Towbin Buffer 58 Towbin Buffer with SDS 58 Transfer 6 Fluorescent Protein Stains 31 Stain-Free Technology 32 Bjerrum Schafer-Nielsen Buffer 58 Detection 6 Colloidal Gold 32 Bjerrum Schafer-Nielsen Buffer with SDS 58 CAPS Buffer 58 General Considerations and Workflow 6 Immunodetection 32 Dunn Carbonate Buffer 58 Immunodetection Workflow 33 0.7% Acetic Acid 58 Chapter 2 Methods and Instrumentation 9 Blocking 33 Protein Blotting Methods 10 Antibody Incubations 33 Detection Buffer Formulations 58 Electrophoretic Transfer 10 Washes 33 General Detection Buffers 58 Tank Blotting 10 Antibody Selection and Dilution 34 Total Protein Staining Buffers and Solutions 59 Semi-Dry Blotting 11 Primary Antibodies 34 Substrate Buffers and Solutions 60 Microfiltration (Dot Blotting) Species-Specific Secondary Antibodies 34 Stripping Buffer 60 Antibody-Specific Ligands 34 Blotting Systems and Power Supplies 12 Detection Methods 35 Tank Blotting Cells 12 Colorimetric Detection 36 Part 3 Troubleshooting 63 Mini Trans-Blot® Cell and Criterion™ Blotter 12 Premixed and Individual Colorimetric Substrates 38 Transfer 64 Trans-Blot® Cell 12 Immun-Blot® Assay Kits 38 Electrophoretic Transfer 64 Trans-Blot® Plus Cell 13 Immun-Blot Amplified AP Kit 38 Microfiltration 65 Semi-Dry Blotting Cells