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 ...

Berkeley, MCB 102

Excerpt: ... nds) Column Chromatography: Principles Ion-exchange chromatography Size-exclusion chromatography Affinity chromatography Specific activity= enzyme activity / amount of protein (mg) Electrophoresis polyacrylamide gel electrophoresis (PAGE) Purification steps Separates charge-to-mass ratio * * Stained with Coomassie * Determining Protein size Molecular weight of protein = daltons Isoelectric focusing electrophoresis (Depends on pI of protein) Two-dimensional electrophoresis Typical 2-D gel ...

Missouri S&T, BIO 391

Excerpt: ... Chapter 11 Structural components How are non-structural viral components defined? What factors make this distinction difficult? List the practical uses for separating structural and non-structural components of a virus. What are the basic principles behind gradient centrifugation? How is gradient centrifugation used to study viruses? Describe SDS polyacrylamide gel electrophoresis (SDS-PAGE). What information we derive from SDS-PAGE? What factors influence the accuracy of that information? Compare the two major methods for DNA sequencing. Describe several physical methods for analyzing genome size. How can nucleic acid hybridization be used to characterize genome size and complexity? Describe how PCR works and how it can be used for analysis of viruses. ...

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 ...

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 ...

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 ...

U. Houston, BCHS 3304

Excerpt: ... id, 20 mM EDTA) Tris MW=121.4 g/mole Boric Acid MW=61.84 g/mole EDTA MW=292.2 g/mole B. 50x TAE (2 M Tris, 2 M Acetic acid, 0.5 M EDTA) Tris MW=121.4 g/mole Concentrated Acetic acid (glacial) comes as a solution and is 17.4 M EDTA MW=292.2 g/mole 2. Your polyacrylamide gel electrophoresis experiment requires 1.0 L of 0.5x TBE. Calculate and describe how you would make this solution using your stock solutions. 3. Your agarose gel electrophoresis experiment requires 500 ml of 1x TAE. Calculate and describe how you would make this solution using your stock solutions. 4. Draw the structure for Methionine. What is its three-letter code? What is its one-letter code? 5. Draw the structure for Cysteine. What is its three-letter code? What is its one-letter code? 6. Complete the following problems from Chapter 4 (p. 40-44) in the Student Companion to Biochemistry: Problems # 2, 5, 6, 7, 8, 10, 13, 14, 18. 7. Complete the following problems from Chapter 4 in (p. 92-93) your Biochemistry textbook: Problems # 1, 3, 6, 7, ...

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 ...

Michigan State University, MMG 451

Excerpt: ... Content List for Chapter 6 Affinity k1: forward or association rate constant k-1: reverse or dissociation rate constant Ka: equilibrium constant for association or association constant Kd: equilibrium constant for dissociation or dissociation constant Equilibrium dialysis Scatchard equation o Scatchard plots o Slope=-Ka o X-axis intercept = n = valence of Ab o Linear vs. curved lines Avidity IgM: avidity compensates for low affinity Cooperativity Cross-reactivity ABO blood types Precipitin Reactions Bivalence or polyvalence of Ab and Ag Equivalence zone Immunoprecipitation Collection of the Ag-Ab complex: secondary Ab, precipitin, protein A, magnetic beads Analysis of labeled proteins by polyacrylamide gel electrophoresis and autoradiography Analysis of unlabeled proteins by Western blot Western blot Polyacrylamide gel electrophoresis SDS Membrane transfer Detection with radiolabeled or enzyme-conjugated Ab Agglutination Agglutinins Prozone effect Incomplete antibodies Passive agglutination Agglutination inhi ...

Mich Tech, BL 4010

Excerpt: ... he universal genetic code used by living systems. (Use diagrams in your answer). C. What did Watson and Crick discover about DNA from its X-ray structure in the 1950s? D. Plants provide carbon and nitrogen compounds (food, vitamins, essential fatty acids) to most other organisms due to their autotrophic nature via photosynthesis, but what else do they do for humans? 3. 20 Points - Protein Purification (5 points each short answer) (Explain your answer briefly but fully use diagrams wherever possible) A. What is native polyacrylamide gel electrophoresis (Native PAGE) used for in protein purifications and how you figure out what the results mean? B. Explain in complete detail how denaturing polyacrylamide gel electrophoresis (SDS-PAGE) works and what can you learn about a protein with this method. C. Explain how one does affinity chromatography for purification of proteins and describe the property of the protein/enzyme you use to do this method. D. Explain how one obtains the subunit composition of a pro ...