MCB 252 Topic 2 Techniques and Technology MCB 252 Summer 2014 Reading for Topic 2 Protein Purification pp 97 99 X ray Crystallography pp 104 5 Antibodies pp 402 404 Microscopy section 9 2 pp 404 411 Transfection Transformation and Protein Tagging pp 203 205 Three Levels of Science 1 What we know memorizing facts Classes an information dump 2 How we know what we know 3 Asking new questions seeking answers making new hypotheses and testing them Topic Outline TechnologyProtein purification X ray crystallography Antibodies Immunofluorescence Transformation Transfection Transgenics GFP How do we identify and study the proteins involved in any particular process How do we know which proteins are produced in which cell types How do we know where any given protein is located within a cell Strategies to study the proteins that are involved in a particular process Biochemistry Purify the proteins involved and study their behavior in vitro Recapitulate the process in a test tube Genetics Isolate a mutant that is defective in the process Cell Biology Watch the process in a microscope Follow the dynamics of particular proteins in a living cell Does inhibiting the function of a specific protein in a living cell block the process you are interested in None of these fields are independent Cell biologists do biochemistry Cell biologists use genetics Geneticists use cell biology Basic Techniques for Studying Biology at the Cell and Molecular Level Biochemistry Protein How do we purify a protein of interest Cell Extract Fractionation 1000 s of Types of proteins Many many copies of each type of protein Separate by Physical Chemical properties Fraction A Fraction B Fraction C Distinct Subsets of Proteins Standard Column Chromatography Protein Elution via Salt in Buffer How do you know which fraction s contain the protein you are interested in In vitro assay How was RNA polymerase II purified In vitro Assay DNA plasmid protein fraction radioactive nts Fractions containing RNAP make RNA polymers Fractions lacking RNAP can t make RNA polymers Radioactivity in RNA polymer precipitates in the presence of TCA Radioactivity nts does not precipitate in the presence of TCA Repeated rounds of purification in combination with an assay allows purification of specific proteins of interest Knowing a Proteins Structure Often Provides Insight into It s Mechanism of Action Chaperonin Protein Folding Device A Molecular Container Knowing a Proteins Structure Often Provides Insight into It s Mechanism of Action DNA Helicase a Molecular Machine Having Purified a Protein How Do We Determine its Structure X ray Crystallography Crystallography does not give us a snapshot of a protein it gives us an electron density map A probability map of where the electrons are most frequently located You cannot determine the structure of the protein without knowing it s amino acid sequence The known size and shape of the amino acids is optimally fit to the electron density map to give us the structure of the protein of interest Must Know Sequence of Protein or DNA to Fit to the Electron Density Map Richmond Fig 4 Cell Biologists often wish to visualize the localization or dynamic properties of a particular protein within a cell One way to do this is immunofluorescence Fluorescence Microscopy Only See What You are Interested in Everything Else is Black Unseen Immunofluorescence Visualizing one specific protein in a cell that contains thousands of proteins 1 We will add a reagent that binds only to the one protein we are interested in What is this reagent An antibody How do we make antibodies 2 That reagent will contain a fluorescent chemical group that will allow us to light up the protein we are interested in How is the chemical group lit up 3 How do we visualize our lit up protein What type of microscope do we need to use Making Antibodies 1 Antigen Inject pure or Enriched protein fraction less than needed For sequencing Antigen Antibodies Ab can be purified from blood Purified Ab s bind antigen Ag specifically in vitro Immune response B Cells Destroyed by Immune System Making Antibodies 2 Polyclonals Antigen B Cell 1 Ab s purified from live animals contain a mixture of Ab s Polyclonal Ab s B Cell 2 B Cell 3 B Cell 4 These Ab s each recognize a different Epitope Making Antibodies 3 Monoclonals B Cell Immortal B Cell Line Cell Fusion B Cell Tissue culture grow in lab Dead Immortal B Cell Line Immortal Cell that makes Ab Tissue culture Alive Permanent continuous source of Ab to a single epitope Monoclonal Ab Making Antibodies 4 Gene Sequence Peptide Synthesis Antibody Antibody Terms Polyclonal Ab Monoclonal Ab Primary Ab Secondary Ab Antigen Epitope Immunofluorescence Visualizing one specific protein in a cell that contains thousands of proteins 1 We will add a reagent that binds only to the one protein we are interested in What is this reagent An antibody How do we make antibodies 2 That reagent will contain a fluorescent chemical group that will allow us to light up the protein we are interested in How is the chemical group lit up 3 How do we visualize our lit up protein What type of microscope do we need to use Light Microscopy Fluorescence Microscopy Visualize Microtubules by Immuno Fluorescense and DNA by a Fluorescent dye DAPI Super impose two pictures taken with two different filters two different wave lengths using a single microscope Comments on Technology Some antibodies bind proteins in such a way that they do not inhibit the function of that protein others bind in such a way that they do inhibit the function of the protein Antibodies that inhibit the function of a protein are often referred to as blocking antibodies Outline TechnologyProtein purification X ray crystallography Antibodies Immunofluorescence Transformation Transfection Transgenics GFP How do we get DNA into cells or organisms How do we express cloned genes in cell or organisms Three versions of this technique transfection transformation transgenics These three techniques differ in the fate of the DNA that put into the cells or organism Transfection Transfection transient expression Transformation Transformation stable long term expression stable cell lines Transgenic Animals Integrate a gene into the genome in the gametes of an organism All progeny in subsequent generations will contain that gene in all cells Some uses of Transformation Transfection Transgenics 1 Put mutant forms of genes and consequently proteins into cells 2 Visualize cells and proteins via