Documents about Polyacrylamide Gel

  • 4 Pages

    lec20

    Purdue, LECT 221

    Excerpt: ... 221 Lecture #20 Poly Acrylamide Gel Electrophoresis (PAGE) PAGE is used to separate/analyze both nucleic acids and proteins. The polyacrylamide gel stabilizes zones and acts as molecular sieve. A solution of acrylamide, CH2=CH-CONH2, in buffer is mixed with a small amount of bis-acrylamide (CH2=CH-CONH-CH2-NH-CO-CH=CH2), which acts as crosslinking agent. Polymerization is initiated by a catalyst which produces free radicals. The polymerized product is a crosslinked polymer chain: . . . -CH2-CH-CH2-CH-. . . . CO NH CH2 NH CO CONH2 CONH2 The porosity of the gel is determined by the total acrylamide concentration, while the stiffness and swelling properties (and to a lesser extent porosity) are controlled by the amount of cross-linking agent. The standard recipies for polyacrylamide gel s use ratios of acrylamide:bis-acrylamide ranging from 19:1, 29:1 up to 37.5:1 and total acrylamide content from 3% to 20% depending on the application. . . .CH2-CH-CH2-CH-. . . Protein Electrophoresis SDS-gel electrophoresis At ...

  • 16 Pages

    CHM381F08Lect08

    Creighton, CHM 381

    Excerpt: ... Protein Purification Objectives Students will Name three common techniques to purify proteins Define the basis of separation for several varieties of chromatography Describe the basis for separation of proteins in PAGE Compare total activity to specific activity Notes Chromatography Ion Exchange Size Exclusion aka Gel Filtration Affinity Chromatography Cadillac of purification When it works Need a specific Ligand for protein Of interest Polyacrylamide Gel Electrophoresis (PAGE) Isoelectric Focusing 2D Electrophoresis Total Activity vs Specific Activity ...

  • 5 Pages

    CELL BIO Lecture1_2009slides

    University of Texas, CH 53170

    Excerpt: ... icroscopy Optical Sections visualized by Confocal Fluor. Microscopy Alberts, 9-21 Alberts, 9-22 How to get things into cells Light vs. Electron Microscope Alberts, 9-34 Alberts, 9-42 Alberts, 9-45 Alberts, 9-50 4 SDS- Polyacrylamide gel electrophoresis (SDS-PAGE) Alberts, 8-19 Alberts, 8-17 SDS- Polyacrylamide gel electrophoresis (SDS-PAGE) Alberts, 8-18 Alberts, 8-18 Western Blotting Alberts, 8-20 5 ...

  • 2 Pages

    sp09_outline03

    Minnesota, CBS 4004

    Excerpt: ... te, antibody, ligand for protein of interest SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) Figs. 8-17, 18, 19 Separates proteins based on molecular mass proteins treated with SDS, -mercaptoethanol Proteins moved by electric field toward positively charged electrode Polyacrylamide gel has small pores, acts a molecular sieve Migration of larger proteins is retarded; smaller proteins move faster Stain with protein dyes to visualize proteins A website with a description of SDS-PAGE is: http:/www.bio.davidson.edu/COURSES/GENOMICS/method/SDSPAGE/SDSPAGE.html Two-dimensional gels separate proteins based on 1) charge (isoelectric point) and 2) molecular weight (Fig. 8-22, 23) Antibodies as tools for purifying, localizing proteins Please review antibody structure on the Cell Biology Interactive disc in the Molecular models folder #25.2 antibodies.mov Antibody (immunoglobulin) protein structure (Figs. 25-21,28,31) Producing antibodies for cell biology experiments: Purify protein to homogenei ...

  • 7 Pages

    Spring 2003 Lecture 11 shorter pdf

    Purdue, CHM 333

    Excerpt: ... CHM333 LECTURES 11: 2/10/03 SPRING 2003 Professor Christine Hrycyna PROTEIN PURIFICATION To study a protein in detail, it must be purified protein sequencing gene cloning crystallography antibodies Chromatography separates components of a mixture based upon their relative affinity for two phases one phase is mobile, the other is stationary gel filtration chromatography (molecular sieve) ion exchange chromatography Electrophoresis separates components of a mixture based upon their charge and/or size polyacrylamide gel electrophoresis (PAGE) isoelectric focussing (IEF) The size or molecular weight of a protein is the sum of the masses of its amino acid residues, and is usually expressed in daltons CHROMATOGRAPHY: Gel filtration chromatography also known as molecular sieve chromatography Separates proteins on the basis of SIZE Mobile phase is a buffer in which proteins are soluble Stationary phase is a column of beads containing pores larger than most of the proteins ...

  • 3 Pages

    Lec20

    UC Riverside, BCH 120

    Excerpt: ... e receptors) also heterodimerize. Heterodimerization with a partner that lacks a DNA-binding domain (e.g. Id inhibitor binds MyoD). Dominant-negative effect of the defective partner (i.e. Id). Analysis of protein - DNA interactions in vitro 1. DNase I Footprinting DNA fragment to be analyzed is radiolabeled at one end with 32P (red dot at 5-end). A. DNase I in limiting amounts cuts radiolabeled DNA about once per DNA molecule randomly (arrows). B. If a protein sits on a specific DNA sequence, DNase I will not cut the DNA fragment at those nucleotide positions. C. The cut DNA fragments generated by Dnase I are then analyzed by electrophoresis on a denaturing polyacrylamide gel . This allows to map the location on the DNA of the protein binding site after autoradiography of the gel (visualization of a footprint). C. The cut DNA fragments generated by Dnase I are then analyzed by electrophoresis on a denaturing polyacrylamide gel . Samples containing the DNA-binding protein Undigested DN ...

  • 25 Pages

    Lecture 4 (Sept 5)

    Vanderbilt, BSCI 110B

    Excerpt: ... are proteins studied? Analyzing complex protein mixtures by SDS-PAGE. 1. "Denature" proteins by boiling in SDS and 2-ME 2. Load protein samples to crosslinked polyacrylamide gel 3. Apply strong electric field 4. SDS-coated proteins migrate towards positive pole. Smaller proteins move faster Small proteins can readily pass Larger proteins retarded by cross-linked polymer How are proteins studied? Analyzing complex protein mixtures by SDS-PAGE. 1. "Denature" proteins by boiling in SDS and 2-ME 2. Load protein samples to crosslinked polyacrylamide gel 3. Apply strong electric field 4. SDS-coated proteins migrate towards positive pole 5. Stain with protein dye or antibodies How are proteins studied? Analyzing complex protein mixtures by SDS-PAGE. How are proteins studied? X-rays Diffraction Determining the 3-D structure of a protein by X-ray crystallography. Protein crystal See Fig. 2.33 Karp 3-D protein structure Next lecture. The enzymatic functio ...

  • 21 Pages

    lec01a.ppt [Compatibility Mode]

    UCLA, LIFESCI LS 3

    Excerpt: ... ques commonly used in molecular biology labs Learn how to use bioinformatics tools and databases Answer various questions in biology How many different polypeptides are found in a protein and what are their sizes? How do living organisms respond to a change in environment? How can DNA be used to determine how related species or individuals are? Lab Hemoglobin: 2 !, 2 " ! Lab I: SDS gel electrophoresis " " Use polyacrylamide gel electrophoresis to determine number and size of subunits in a protein Basic technique essential in molecular biology and biochemistry How can we detemine the size and number of polypeptides in a protein? hemoglobin ! Purify protein ! Di Dissociate into separate polypeptides i t i t t l tid (detergent: SDS) ! Separate using polyacrylamide gel electrophoresis SDS Polyacrylamide gel forms matrix that separates p proteins based on size Lab 1 ! You'll be given unknown protein of g given MW " " " Denature Separate subunits on polyacrylamide gel Determine how many of each subu ...

  • 16 Pages

    BBlecture12S06

    W. Alabama, BIOL 240

    Excerpt: ... verage of the genome to ensure most sequence is obtained gaps are filled in manually genomics websites: http:/www.tigr.org http:/www.genomesonline.org Sinorhizobium meliloti genome (see course notes under "Lecture 12" for hot links) open reading frame (orf): sequence of DNA that, if transcribed, could be translated to yield a protein of known length, composition chromosome map of Mycobacterium tuberculosis: size (Mb) ORFs IS, other repetitive DNA elements locations of tRNA genes histogram showing regional G+C content rRNA operon (Madigan & Martinko Fig 15.5) some prokaryotic chromosomes: Haemophilus influenzae Rd 1,830,137 1st cellular chromosome sequenced (1995) in prokaryotes, ORF content is proportional to genome size: overview of metabolic pathways, transport systems in Thermotoga maritima: distribution of gene function types in bacterial genomes: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of E. coli proteins: making and using DNA chips: Saccharom ...

  • 16 Pages

    BBlecture12S06

    W. Alabama, BIOL 240

    Excerpt: ... verage of the genome to ensure most sequence is obtained gaps are filled in manually genomics websites: http:/www.tigr.org http:/www.genomesonline.org Sinorhizobium meliloti genome (see course notes under "Lecture 12" for hot links) open reading frame (orf): sequence of DNA that, if transcribed, could be translated to yield a protein of known length, composition chromosome map of Mycobacterium tuberculosis: size (Mb) ORFs IS, other repetitive DNA elements locations of tRNA genes histogram showing regional G+C content rRNA operon (Madigan & Martinko Fig 15.5) some prokaryotic chromosomes: Haemophilus influenzae Rd sequenced (1995) 1,830,137 1st cellular chromosome in prokaryotes, ORF content is proportional to genome size: overview of metabolic pathways, transport systems in Thermotoga maritima: distribution of gene function types in bacterial genomes: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of E. coli proteins: making and using DNA chips: Saccharom ...

  • 2 Pages

    BIO 320 Lecture1notes_2009

    University of Texas, BIO 50160

    Excerpt: ... BIO320CELLBIOLOGYSpring2009 T.OHalloran&A.DeLozanne Lecture1 MethodsinCellBiology(January20,2009) Reading:Alberts,5thEd.Chapter8,thefollowingsections: Isolatingcellsandgrowingtheminculture Cellscanbegrowninculture Purifyingproteins Cellscanbesep ...

  • 6 Pages

    Gel_Electrophoresis_Procedure

    E. Kentucky, BIOL 659

    Excerpt: ... ound color. Distilled water. 10% glycerol in 20% aqueous EtOH. Polyacrylamide gel s Sample dissociation buffer Gel Staining Solution Destaining Solution Gel Preservative Solution Gel Electrophoresis Procedure Page 49/6 II. PROTOCOL Important: Refer to any additional handouts for the latest revisions to this protocol! This protocol describes analysis of the fractions resulting from column chromatography. Adjust the number of samples as necessary for your particular analysis. Adjust the volume of samples to match your needs and the capacity of the gel. A. Preparing the Sample. This can be done in the lab period before the gels are run. 1. Obtain a number of microcentrifuge tubes for each sample plus two for markers. Label the top of each tube with the number of the fraction or sample, or with "M" for marker proteins (size calibrations standards). 2. Into each tube labeled "M", pipette ca. 3-5 L of the Protein Size Standard (Marker Protein) mix. Calculate the volume so that you have ca. 0.1 to 0.2 g (100- ...

  • 8 Pages

    482lec7

    Mich Tech, BL 4820

    Excerpt: ... BL4820 BASIC BIOCHEMICAL TECHNIQUES Lecture 7 - Polyacrylamide Gel Electrophoresis (PAGE) on GOT - Expt 4 Part A Read in Text: p. 43-45; 48-49 Polyacrylamide Gel Electrophoresis (PAGE) for Proteins There are two types of PAGE which you will do: 1) Native PAGE - in Week 7 2) SDS-PAGE (Denaturing PAGE) - in Week 8 1. Native PAGE PAGE is probably the most highly resolving electrophoretic method yet developed for separating proteins. In electrophoresis, proteins are separated by charge-density. In gel electrophoresis, the support media (the gel) also contributes to the separation power of the method. Using the organic monomer, acrylamide, to make the gel by free radical polymerization results in very uniform pore sizes which can be reproduced each time a gel is poured without a lot of variation. How to make a PAGE Gel. Chemistry: Since the pores in a PAGE gel are the size of proteins, molecular sieving contributes to the resolving power of PAGE. Consequently, PAGE is a high resolution method and ...

  • 1 Pages

    abstracts_4963

    Cornell, ARP 38

    Excerpt: ... Program # 626.7 Regulation of Protein Expression in Escherichia coli by Autoinducer-2 Adam Robert Parks1, Martha Stapels2, Jeffrey B Schineller1, Douglas F Barofsky2. 1Chemistry, Humboldt State University, 1 Harpst St., Arcata, CA 95521-8299, 2Chemistry, Oregon State University, Corvallis, OR Escherichia coli regulates the expression of specific genes in response to signal molecules present in their environment. These signal molecules, termed autoinducers, increase in concentration proportionally with cell density during growth. In this study, we used two methods to observe the changes that take place in the proteome of E. coli following induction by autoinducer-2 (AI-2). Both Isotope Coded Affinity Tags (ICAT) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used in conjunction with mass spectrometry to identify proteins from bulk E. coli cell lysates. The differentially expressed proteins that we observed indicate a shift in nutrient utilization, conservation of existing resources, a ...

  • 82 Pages

    Electrophoresis

    Georgia Tech, CHEM 4581

    Excerpt: ... tech nical manual protein electrophoresis Protein Electrophoresis technical manual tm 80-6013-88/Rev. B0/12-99 page nder Page nder This technical manual contains information that has been distilled from more than 30 years of laboratory experience in developing and verifying applications and product performance for protein electrophoresis. As such it is ideal for both new and current users of protein electrophoresis as both a teaching and a reference guide. The rst chapter provides a theoretical framework, and Chapters 2 through 4 cover the major aspects of protein electrophoretic separation: polyacrylamide gel electrophoresis, isoelectric focusing, and gel analysis. In addition, an extensive reference list is summarized at the end of every chapter, and a detailed glossary is included at the end of the manual. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Chapter 1 Introduction to Electrophoretic Theory . . . . . . . ...

  • 19 Pages

    lecture12

    W. Alabama, BIOL 240

    Excerpt: ... verage of the genome to ensure most sequence is obtained gaps are filled in manually genomics websites: http:/www.tigr.org http:/www.genomesonline.org Sinorhizobium meliloti genome (see course notes under "Lecture 12" for hot links) open reading frame (orf): sequence of DNA that, if transcribed, could be translated to yield a protein of known length, composition chromosome map of Mycobacterium tuberculosis: size (Mb) ORFs IS, other repetitive DNA elements locations of tRNA genes histogram showing regional G+C content rRNA operon some prokaryotic chromosomes: in prokaryotes, ORF content is proportional to genome size: overview of metabolic pathways, transport systems in Thermotoga maritima: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of E. coli proteins: making and using DNA chips: Saccharomyces cerevisiae genome affixed to silica chip: probed with fluorescently labelled mRNA from yeast cells grown under specific conditions non-blue colour shows DNA-RNA ...

  • 19 Pages

    lecture12

    W. Alabama, BIOL 240

    Excerpt: ... verage of the genome to ensure most sequence is obtained gaps are filled in manually genomics websites: http:/www.tigr.org http:/www.genomesonline.org Sinorhizobium meliloti genome (see course notes under "Lecture 12" for hot links) open reading frame (orf): sequence of DNA that, if transcribed, could be translated to yield a protein of known length, composition chromosome map of Mycobacterium tuberculosis: size (Mb) ORFs IS, other repetitive DNA elements locations of tRNA genes histogram showing regional G+C content rRNA operon some prokaryotic chromosomes: in prokaryotes, ORF content is proportional to genome size: overview of metabolic pathways, transport systems in Thermotoga maritima: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of E. coli proteins: making and using DNA chips: Saccharomyces cerevisiae genome affixed to silica chip: probed with fluorescently labelled mRNA from yeast cells grown under specific conditions non-blue colour shows DNA-RNA ...