DOCSLIB.ORG

Bioanalytical Chemistry 8. Gel Electrophoresis and Blotting

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bioanalytical Chemistry 8. Gel Electrophoresis and Blotting 91 Bioanalytical chemistry 8. Gel electrophoresis and blotting Suggested 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 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/. • Cancer examples from Weinberg, Robert (2007). The Biology of Cancer. Garland Science. • http://www.piercenet.com/ Electrophoresis 92 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). Ez v = f The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity (η) of the medium. For a sphere of radius r, f = 6πηr € The speed of migration is therefore proportional to the charge:mass ratio. z Will the charge to mass ratio differ v ∝ between proteins? Between r different DNA molecules? € • Electrophoresis is a technique for separating, or resolving, molecules in a mixture under the influence of an applied electric field. Dissolved molecules in an electric field move, or migrate, at a speed determined by their charge:mass ratio. • For example, if two molecules have the same mass and shape, the one with the greater net charge will move faster toward an electrode. € Agarose gel electrophoresis is typically used 93 for the separation of DNA molecules • Agarose is a polysaccharide that is obtained from the cell wall of some seaweed species. • The basic repeating unit of agarose is 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α-L- galactopyranose. Two units of this repeating structure, known as agarobiose, are shown on the slide. • The average molecular mass of the agarose polysaccharide is ~120,000 daltons, which corresponds to ~400 agarobiose units. Some of the sugar groups in the polysaccharide are sulfated. • Agarose is essentially insoluble at room temperature but can be dissolved in water at high temperature. By heating and cooling a solution of agarose, a firm gel can be formed. The pores within this gel depend on the concentration of agarose used, but can be in the range of 100 to 300 nm. This pore size is sufficient to allow DNA molecules to pass through in an applied electric field. However, there is very little passive diffusion of DNA through the gel. • The mechanism for gelation of agarose upon cooling is thought to involves the formation of double helix structures in the initial stages of gelation. These double helixes then pack together into bundles to give the final gel structure. • Some information and the gelation mechanism figure from www.Lonza.com (MB_SB_appendixB_207-209.pdf) • What is agar and agarose? See http://www.lsbu.ac.uk/water/hyagar.html DNA is typically fragmented with restriction 94 enzymes prior to electrophoresis • this is the traditional method for paternity testing and identifications based on blood left at a crime scene • a standard set of restriction enzymes is chosen such that each person has a unique ‘signature’ pattern of bands. • common SNPs could occur at a particular restriction site, or perhaps there are restriction sites that flank a region of chromosome that is highly variable in length image credit: U.S. Department of Energy Human Genome Program, http://www.ornl.gov/hgmis. • Gel electrophoresis is used to separate DNA and RNA molecules by size and to estimate the size of nucleic acid molecules of unknown length by comparison with the migration of molecules of known length. • DNA and RNA molecules are highly charged near neutral pH because the phosphate group in each nucleotide contributes one negative charge. As a result, DNA and RNA molecules move toward the positive electrode during gel electrophoresis. Smaller molecules move through the gel matrix more readily than larger molecules, so that molecules of different length, such as restriction fragments, separate. Because the gel matrix restricts random diffusion of the molecules, molecules of different length separate into “bands” whose width equals that of the well into which the original DNA mixture was placed. Pulsed field agarose gel electrophoresis can 95 separate very large DNA molecules Normal mode Pulsed field Recall that the smallest human chromosome is ~50 million base pairs. • Agarose gel electrophoresis with a constant electric field can resolve DNA fragments up to ≈20 kb in length. Larger DNAs, ranging from 20 kb to 10 megabases (Mb) in length, can be separated by size with pulsed-field gel electrophoresis. • This technique depends on the unique behaviour of large DNAs in an electric field that is turned on and off (pulsed) at short intervals. When an electric field is applied to large DNA molecules in a gel, the molecules migrate in the direction of the field and also stretch out lengthwise. If the current then is stopped, the molecules begin to “relax” into random coils. The time required for relaxation is directly proportional to the length of a molecule. • The electric field then is reapplied at 90° or 180° to the first direction. Longer molecules relax less than shorter ones during the time the current is turned off. Since the molecules must relax into a random coil before moving off in a new direction, longer molecules start moving in the direction imposed by the new field more slowly than shorter ones. Repeated alternation of the field direction gradually forces large DNA molecules of different size farther and farther apart. • Pulsed-field gel electrophoresis is very important for purifying long DNA molecules up to ≈107 base pairs in length. Very large chromosomes must be digested into fragments of 107 base pairs or less before they can be analyzed. Such large restriction fragments can be generated with restriction enzymes that cut at rarely occurring 8-bp restriction sites. DNA fragments can be detected by 96 autoradiography or fluorescent dyes Detection of DNA fragments in gels: 1. Radioactive label + photographic film or a phosphorimager 2. Fluorescent dye a. Ethidium bromide Ethidium b. SYBR or monomeric cyanine dyes Yo-pro-1 c. Dimeric cyanine dyes %% Yoyo-1 http://www.probes.com/handbook/sections/0804.html • Whereas agarose gel electrophoresis is typically used to resolve DNA molecules that differ by at least ~100 base paris or so, the resolving power of polyacrylamide gel electrophoresis is so great that single-stranded DNA molecules up to about 500 nucleotides long can be separated if they differ in length by only 1 nucleotide. This high resolution is critical to the DNA- sequencing procedures described later. • DNA molecules composed of up to ≈2000 nucleotides can be separated electrophoretically on polyacrylamide gels, and molecules from 500 nucleotides to 20 kb on agarose gels. • 3 gels labeled A to C: (A) a polyacrylamide gel is used to fractionate single-stranded DNA. In the size range 50 to 200 nucleotides, DNA molecules that differ in size by only a single nucleotide can be separated from each other. (B) an agarose gel with medium-sized pores is used to separate double-stranded DNA molecules. This method is most useful in the size range 300 to 10,000 nucleotide pairs. (C) the technique of pulsed-field agarose gel electrophoresis has been used to separate 16 different chromosomes that range in size from 220,000 to 2.5 million nucleotide pairs. DNA molecules as large as 107 nucleotide pairs can be separated in this way. • Radioactively labeled DNA can be visualized by autoradiography of the gel. In this case, the gel is laid against a sheet of photographic film in the dark, exposing the film at the positions where labeled DNA is present. When the film is developed, a photographic image of the DNA is observed. Radiolabeled DNA bands also can be detected by laying the gel against a phosphorimager screen, which counts β-particles released by labeled molecules in the gel. • If the DNA is not radiolabeled, the gel is incubated in a solution containing the fluorescent dye ethidium: This planar molecule binds to DNA by intercalating between the base pairs. Binding concentrates ethidium in the DNA and also increases its intrinsic fluorescence. As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regions of the gel without DNA. • Q: On diagram C the numbers that are on the left that are not in an order what are those refering to? • A: Those are chromosome numbers. This DNA must be from a species that has much smaller chromosomes than humans. 97 Analyzing very complex mixtures of genomic DNA: the Southern blot • "Gels used for electrophoresis of nucleic acids and proteins are permeable. This obvious fact didn't dawn on me until I tried to dissolve some agarose by floating it on a solution of sodium perchlorate and noticed a bead of liquid form on the top. I reasoned that if DNA molecules were carried through with the flow it would be possible to capture them on a nitrocellulose membrane, using the setup shown in the sketch. The big thrill came when, at the first attempt, I saw genes lit up as bands after hybridizing with radiolabeled probes.
Load more

Recommended publications
  • Control of Molecular Motor Motility in Electrical Devices
    Control of Molecular Motor Motility in Electrical Devices Thesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor in Philosophy By Laurence Charles Ramsey Department of Electrical Engineering & Electronics April 2014 i Abstract In the last decade there has been increased interest in the study of molecular motors. Motor proteins in particular have gained a large following due to their high efficiency of force generation and the ability to incorporate the motors into linear device designs. Much of the recent research centres on using these protein systems to transport cargo around the surface of a device. The studies carried out in this thesis aim to investigate the use of molecular motors in lab- on-a-chip devices. Two distinct motor protein systems are used to show the viability of utilising these nanoscale machines as a highly specific and controllable method of transporting molecules around the surface of a lab-on-a-chip device. Improved reaction kinetics and increased detection sensitivity are just two advantages that could be achieved if a motor protein system could be incorporated and appropriately controlled within a device such as an immunoassay or microarray technologies. The first study focuses on the motor protein system Kinesin. This highly processive motor is able to propel microtubules across a surface and has shown promise as an in vitro nanoscale transport system. A novel device design is presented where the motility of microtubules is controlled using the combination of a structured surface and a thermoresponsive polymer. Both topographic confinement of the motility and the creation of localised ‘gates’ are used to show a method for the control and guidance of microtubules.
    [Show full text]
  • Molecular Biologist's Guide to Proteomics
    Molecular Biologist's Guide to Proteomics Paul R. Graves and Timothy A. J. Haystead Microbiol. Mol. Biol. Rev. 2002, 66(1):39. DOI: 10.1128/MMBR.66.1.39-63.2002. Downloaded from Updated information and services can be found at: http://mmbr.asm.org/content/66/1/39 These include: REFERENCES This article cites 172 articles, 34 of which can be accessed free http://mmbr.asm.org/ at: http://mmbr.asm.org/content/66/1/39#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on November 20, 2014 by UNIV OF KENTUCKY Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2002, p. 39–63 Vol. 66, No. 1 1092-2172/02/$04.00ϩ0 DOI: 10.1128/MMBR.66.1.39–63.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Molecular Biologist’s Guide to Proteomics Paul R. Graves1 and Timothy A. J. Haystead1,2* Department of Pharmacology and Cancer Biology, Duke University,1 and Serenex Inc.,2 Durham, North Carolina 27710 INTRODUCTION .........................................................................................................................................................40 Definitions..................................................................................................................................................................40 Downloaded from Proteomics Origins ...................................................................................................................................................40
    [Show full text]
  • Protein Facility
    Supporting core service facilities for biotechnology research by faculty, student, government, and industry scientists. More Office of Biotechnology at www.biotech.iastate.edu/service_facilities. Protein Facility Iowa State University’s Protein purification of proteins and of protein samples from 1D or 2D Facility provides expertise for the peptides can be accomplished. gels. Gel spots can be digested analysis, characterization, and with a variety of enzymes, and synthesis of proteins and peptides. Mass Spectrometry the resulting peptides can be After training, users can operate A matrix-assisted laser desorption/ analyzed to identify the protein. many instruments themselves. ionization time-of-flight (MALDI- TOF) mass spectrometer can be used SDS-PAGE / Electroblotting for determining the molecular weight The facility conducts sodium of proteins, peptides, glycoproteins, dodecyl sulfate polyacrylamide oligosaccharides, oligonucleotides, gel electrophoresis (SDS-PAGE) of and polymers. A quadrapole- proteins for purity and molecular TOF tandem mass spectrometer weight estimation. Gels can be is also available for obtaining electroblotted to nitrocellulose peptide sequence information. or to PVDF for immuno- detection and protein/peptide Peptide Synthesis sequencing, respectively. The facility can do both large- and small-scale peptide synthesis, 2-D Gel Electrophoresis including phosphopeptides, peptides The facility does two-dimensional with unusual amino acids, and electrophoresis by separating proteins multiple antigenic peptides (MAP). in the first dimension according to charge (isoelectric focusing), Circular Dichroism Protein and Peptide Sequencing followed by separating the focused Researchers who want to detect The Protein Facility provides proteins in the second dimension and quantitate the chirality of N-terminal protein/peptide sequence according to their molecular weight.
    [Show full text]
  • Impact Factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 REVIEW ON: ELECTROPHORESIS: METHOD for PROTEIN SEPARATION Shindedipa
    Impact factor: 3.958/ICV: 4.10 ISSN: 0978-7908 192 Pharma Science Monitor 7(2),Apr-Jun 2016 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com REVIEW ON: ELECTROPHORESIS: METHOD FOR PROTEIN SEPARATION ShindeDipa V.*, JasminaSurati Department of Quality Assurance, Shree NaranjibhaiLalbhai Patel College of Pharmacy,Umrakh -394 345,Bardoli, Gujarat, India. ABSTRACT Electrophoresis is one of the widely used techniques in molecular biochemistry, microbiology, biomedical research. It is a type of protein separation method .It is one of the highly efficient techniques of analysis and sole method for separation of proteins for western blot, RNA studies etc. It is a both qualitative and quantitative analysis technique. Separation depend upon electrophoretic mobility.Electrophoresis technique are of various type like Moving boundary electrophoresis ,Zone electrophoresis ,Affinity electrophoresis ,Pulsed field electrophoresis ,Dielectrophoresis.this technique mainly used in antibiotic analysis,vaccine analysis DNA analysis and protein analysis as well as fingerprint analysis. KEYWORDS:Electrophoresis, Electrophoretic mobility,Zone Electrophoresis, Moving boundary Electrophoresis, Dielectricphoresis. INTRODUCTION Electrophoresis is a physical method of analysis based on the migration of electrically charged proteins, colloids, molecules or other particles dissolved or dispersed in an electrolyte solution in the direction of the electrode bearing the opposite polarity when an electric current is passed through it. Separations may be conducted in systems without support phases (such as free solution separation in capillary electrophoresis) or in stabilising media such as thin-later plates, filins or gels. The electrophoretic mobility is the rate of movement in metres per second of the charged particles under the action of an electric field of I volt per metre and is expressed in square metres per volt second.
    [Show full text]
  • Permease from Escherichia Coli
    Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli Mikhail Bogdanova,1, Philip Heacocka, Ziqiang Guanb, and William Dowhana,1 aDepartment of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX, 77030; and bDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710 Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved July 15, 2010 (received for review May 9, 2010) Phosphatidylcholine (PC) has been widely used in place of naturally N-terminal two-TM helical hairpin of PheP and GabP are in- occurring phosphatidylethanolamine (PE) in reconstitution of verted with respect to their orientation in PE-containing cells bacterial membrane proteins. However, PC does not support native and the remaining TMs. These permeases do not carry out pro- structure or function for several reconstituted transport proteins. ton-coupled energy-dependent uphill transport of substrate in Lactose permease (LacY) of Escherichia coli, when reconstituted cells lacking PE, but still display energy-independent downhill in E. coli phospholipids, exhibits energy-dependent uphill and transport. LacY reconstituted into total E. coli phospholipids car- energy-independent downhill transport function and proper con- ries out uphill transport with domains C6 and P7 on opposite formation of periplasmic domain P7, which is tightly linked to sides of the membrane bilayer as observed in wild-type cells uphill transport function. LacY expressed in cells lacking PE and (9). Leaving out PE during reconstitution results in only downhill containing only anionic phospholipids exhibits only downhill trans- transport with domains C6 and P7 residing on the same side of port and lacks native P7 conformation.
    [Show full text]
  • 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
    [Show full text]
  • Wave Electroblotting, 2D, Complete Electrophoresis Systems And
    Instruction Manual WAVE Standard and Tetrad Electroblotting, 2-D and Complete Electrophoresis Systems Electroblotting VS20WAVECBS AND WAVETETRAD1CBS Includes VS20WAVESYS and WAVEBI SW20 & WAVEBI 2-D Electrophoresis WAVEC2DS Includes VS20WAVESYS and VS20WAVEDCI VS20WAVEDC Complete (Electroblotting & 2-D Electrophoresis) VS20WAVECES AND WAVETETRAD1CES Includes VS20WAVECBS and VS20WAVEDCI 1 Contents:- Page 1) Safety Instructions 3 2) Packing Lists 4 3) Care and Maintenance 6 4) Usage Guidance and restrictions: 7 5) Set Up 8 6) Gel Casting 13 7) Gel Preparation 20 8) Gel Selection 20 9) Gel Pouring 22 10) Sample Preparation and Loading 23 11) Buffer Volume 25 12) Gel Running 25 13) Solutions 26 14) Vertical Gel Electrophoresis References 28 15) Combs 29 16) Protein Blotting using the WAVE 31 17) Buffer Volumes 32 18) Passive and Active Cooling 32 19) Run Conditions 34 20) Blotting References 35 21) Buffers 36 22) Blotting Troubleshooting 37 23) 1st Dimension Electrophoresis using the WAVEDCI Tube Gel Module 41 24) Capillary Tube Gel Pouring 41 25) 1st Dimension (IEF) Phase Tube Gel Running 43 26) 2-D, Size Determination Phase 44 27) Warranty 45 2 SAFETY PRECAUTION WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK. HOWEVER, THESE UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY AND ARE TO BE OPERATED ONLY BY QUALIFIED PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN THIS INSTRUCTION MANUAL. ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE MANUAL THOROUGHLY. THE UNIT MUST NEVER BE USED WITHOUT THE SAFETY LID CORRECTLY IN POSITION. THE UNIT SHOULD NOT BE USED IF THERE IS ANY SIGN OF DAMAGE TO THE EXTERNAL TANK OR LID.
    [Show full text]
  • Power and Limitations of Electrophoretic Separations in Proteomics Strategies
    Power and limitations of electrophoretic separations in proteomics strategies Thierry. Rabilloud 1,2, Ali R.Vaezzadeh 3 , Noelle Potier 4, Cécile Lelong1,5, Emmanuelle Leize-Wagner 4, Mireille Chevallet 1,2 1: CEA, IRTSV, LBBSI, 38054 GRENOBLE, France. 2: CNRS, UMR 5092, Biochimie et Biophysique des Systèmes Intégrés, Grenoble France 3: Biomedical Proteomics Research Group, Central Clinical Chemistry Laboratory, Geneva University Hospitals, Geneva, Switzerland 4: CNRS, UMR 7177. Institut de Chime de Strasbourg, Strasbourg, France 5: Université Joseph Fourier, Grenoble France Correspondence : Thierry Rabilloud, iRTSV/LBBSI, UMR CNRS 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr Abstract: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry( i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed. Keywords: electrophoresis, two-dimensional electrophoresis, isoelectric focusing, immobilized pH gradients, peptides, proteins, proteomics. Table of contents I. Introduction II. The principles at play III. How to use electrophoresis in a proteomics strategy III.A.
    [Show full text]
  • Electroblot Proteins by Wet Or Semi-Dry Transfer
    TECH TIP #73 Electroblot proteins by wet or semi -dry transfer TR0073.1 Introduction Typical Western blotting experiments involve polyacrylamide gel electrophoresis (PAGE) of protein samples followed by transfer of the size-separated proteins from gel to nitrocellulose or PVDF membrane. Bands or spots of specific proteins are then visualized on the membrane surface when probed with antibodies and detected with enzyme-substrate reporter systems. Three different methods can be used for protein transfer: passive-diffusion blotting, vacuum blotting, and electroblotting. Among these methods, electroblotting is the most popular because it is both faster and more efficient than the others. Electrophoretic transfer also is more quantitative. Proteins are transferred by an electric current passed through the gel. Wet (tank) transfer and semi-dry transfer have been developed to electrophoretically blot proteins and nucleic acids from gels to membranes. Transfer efficiency in these two variants of electroblotting is dependant upon gel type, membrane type, transfer buffer composition, equilibration time, size of protein(s), transfer temperature, number of gels, and volume of buffer. The purpose of this Tech Tip is to provide very simple, generalized protocols for wet and semi-dry transfer of proteins from standard mini gels (approx. 10cm × 10cm) using typical commercial transfer devices and power supplies. When possible consult and follow the instructions for the specific equipment you own. Relevant Thermo Scientific™ Pierce™ products are mentioned in each protocol, and a fuller list of these products occurs at the end this document. Protein blotting using a wet (tank) transfer apparatus 1. Separate the proteins in the sample by gel electrophoresis (e.g., reducing, denaturing SDS-PAGE).
    [Show full text]
  • Quality Control of Overexpressed Membrane Proteins
    Quality control of overexpressed membrane proteins Eric R. Geertsma, Maarten Groeneveld, Dirk-Jan Slotboom, and Bert Poolman* Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Communicated by H. Ronald Kaback, University of California, Los Angeles, CA, March 4, 2008 (received for review January 21, 2008) Overexpression of membrane proteins in Escherichia coli fre- optimization process. Ideally, the quantification should avoid quently leads to the formation of aggregates or inclusion bodies, extra experimental steps, which would prevent high-throughput which is undesirable for most studies. Ideally, one would like to analysis, and should not involve relating different types of data, optimize the expression conditions by monitoring simultaneously which would make comparisons cumbersome. and rapidly both the amounts of properly folded and aggregated Here, we present an application based on the use of GFP as membrane protein, a requirement not met by any of the currently protein folding indicator, in which not only the folded protein is available methods. Here, we describe a simple gel-based approach quantified, but also the nonfolded protein. The method is based with green fluorescent protein as folding indicator to detect well on differential migration of folded and aggregated GFP fusion folded and aggregated proteins simultaneously. The method al- proteins during SDS/PAGE. Subsequent immunodetection of lows for rapid screening and, importantly, pinpointing the most both species allows simultaneous determination of the levels of likely bottlenecks in protein production. folded and aggregated protein present. This additional informa- tion greatly accelerates the optimization of the functional over- folding indicator ͉ in gel GFP fluorescence ͉ optimization of expression of both membrane and soluble proteins.
    [Show full text]
  • Intradimerically Tethered DNA Topoisomerase II Is Catalytically Active in DNA Transport (Leucine Zipper/Cross-Linking/DNA Gyrase) JANET E
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2975-2980, April 1996 Biochemistry Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport (leucine zipper/cross-linking/DNA gyrase) JANET E. LINDSLEY Department of Biochemistry, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132 Communicated by Nicholas R. Cozzarelli, University of California, Berkeley, CA, December 18, 1995 (receivedfor review August 14, 1995) ABSTRACT A covalently cross-linked dimer ofyeast DNA Two general mechanisms, often termed the "one-gate" and topoisomerase II was created by fusing the enzyme with the "two-gate" models, have been proposed for the functioning of GCN4 leucine zipper followed by two glycines and a cysteine. topoisomerase II (3, 19-22). It is proposed in the one-gate Upon oxidation ofthe chimeric protein, a disulfide bond forms model that all DNA substrates and products enter and exit between the two carboxyl termini, covalently and intradimeri- from the same face of the enzyme. In the two-gate model, the cally cross-linking the two protomers. In addition, all nine of transported duplex enters one face of the enzyme, is passed the cysteines naturally occurring in topoisomerase II have through the cleaved DNA duplex, and exits from the opposite been changed to alanines in this construct. This cross-linked, face. Results of an elegant, although indirect, experiment by cysteine-less topoisomerase II is catalytically active in DNA Roca and Wang (20) support a two-gate mechanism. duplex passage as indicated by ATP-dependent DNA supercoil To further understand the interactions between the two relaxation and kinetoplast DNA decatenation assays.
    [Show full text]
  • Optimized Method for Rapid Protein Electroblotting
    ADVERTISING FEATURE APPLICATION NOTES Optimized method for rapid protein electroblotting Protein electroblotting is a common method for transferring proteins out of a polyacrylamide gel onto a membrane for subsequent western blotting. With a high-current power supply, such as that found in the Thermo Scientific™ Pierce™ G2 Fast Blotter, and a high–ionic strength transfer buffer, protein transfer from nitrocellulose or polyvinylidene difluoride (PVDF) membranes can be achieved in 5–10 m, even with proteins as large as 300 kDa. Introduction Western blotting is a commonly used technique for detection of proteins. The quality of data derived from western blotting is dependent on several factors, including the sample integrity, blocking buffer, antibody quality, antibody concentration, type of membrane and assay technique. One of the most critical factors for satisfactory western blot results is consistent and efficient transfer of the proteins from the gel to the membrane. Traditional transfer techniques include tank and semidry transfers. The Pierce™ G2 Fast Blotter (Fig. 1) represents a semidry transfer method. Tank transfer is often considered the optimal method for electroblotting because it is very reproducible and generally provides good transfer efficiency. In tank transfer, the transfer sandwich is secured in a cassette and submerged in a tank of transfer buffer. This method uses a large volume of buffer containing methanol and © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature is usually performed with an ice pack within the transfer tank or by Figure 1 | The Thermo Scientific™ Pierce™ G2 Fast Blotter can transfer proteins placing the whole apparatus in a cold room to offset the heat that is from gel to membrane in 10 m.
    [Show full text]