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BIOLOGY 2B03 (285)
Kim Dej (39)
Lecture 3

Bio 2B03 Lecture 3.docx

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Kim Dej

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Bio 2B03 Lecture 3 Chapter 3: 3.6- Purifying, Detecting and Characterizing Proteins  Molecules can be separated based on their physical or chemical characteristics  Characteristics used for separation include Size, defined as either length or mass, and binding affinity for several ligands  Radioactive compounds for tracking biological activity Centrifugation:  can separate particles and molecules that differ in mass or density  particles in suspension with different masses or densities will settle to the bottom of the tubes at different rates; heavier particles will settle first Differential Centrifugation:  Mixture (called cell homogenate) is spun at a rotor speed that forces cell organelles such as nuclei and large unbroken cells or large cell fragments to collect as a pellet at the bottom, the soluble proteins remain in the supernatant Liquid Chromatography  Resolves proteins by mass, charge or binding affinity  Based on the principle that molecules dissolved in a solution can differentially interact( bind and dissociate) with a particular solid surface  Frequent interactions will result in molecules that will spend more time bound to the surface and thus flow past the surface more slowly and vice versa  Nature of the beads in the column determines whether the separation of proteins depends on the differences in mass, charge, or binding affinity Gel Filtration Chromatography:  Composed of porous beads made from polyacrylamide, dextran ( a bacterial polysaccharide) or agarose ( a seaweed derivative)  Smaller protein can penetrate into the a bead’s depression more readily than larger proteins (based on size and shape)  Smaller mass = more time trapped in bead Ion-Exchange Chromatography:  separated on the basis of their charges  beads surfaces are covered by either amino groups ( a positive charge- NH3+) or carboxyl groups (a negative charge COO-)  Low salt concentration: molecules and beads are attracted by their opposite charges  High salt concentration: negative salt ions bind to the positively charged beads, displacing the negative charged proteins ( Adding a salt solution will elute the protein that has a different charge from the bead)  Increasing salt concentration will result in the elution of weakly bound proteins (relatively low charge) first and then highly charged proteins Affinity Chromatography:  In this technique, ligand or other molecules that bind to the protein of interest are covalently attached to the beads used to form the column  Attached molecule is an antibody specific for the desired protein  Will retains only those proteins that bind the molecules attached to the beads  Proteins bound are then eluted by adding an excess of a soluble form of the ligand or by changing the salt concentration or pH Electrophoresis:  Separates molecules based on their charge to mass ratio (charge: mass) under the influence of an applied electric field  If two molecules have the same mass and shape, the one with the greater newt charge will move faster towards an electrode of the opposite polarity SDS- Polyacrylamide Gel Electrophoresis (PAGE):  Usually little or no separation due to identical charge: mass ratio  Successful separation of molecules can be accomplished by electrophoresis in gel  Smaller proteins migrate faster through the gel than larger proteins (smaller species proceed faster through the pores in the gel than larger species)  Shape also matters; long asymmetric molecules migrate more slowly than spherical ones of the same mass  Rate at which the protein moves through the gel is influenced by the gel’s pore size and the strength of the electric field  For resolving protein mixtures, proteins are exposed to the ionic detergent SDS (sodium dodecylsulfate) before and during gel electrophoresis  SDS denatures proteins by destabilizing the hydrophobic interactions that contribute to the proteins stable structure  Polypeptide chains are extended into conformations with similar charge: mass ratio, allowing SDS to eliminate the effect of differences in shape in native structures  Linear relationship between migration distance and the log of the molecular weight  SDS-PAGE can resolve proteins having relatively large masses but cannot readily resolve proteins having the same mass  To separate proteins of similar mass, the characteristic “electric charge” is exploited, which is determined by the pH (Two Dimensional Gel Electrophoresis)  In Two Dimensional Gel Electrophoresis, proteins are separated first by their charges and then their mass Chapter 9: 9.2- Light Microscopy: Visualizing Cell Structure and Localizing Proteins within Cells  Light microscopes enable biologists to reveal the myriad movements of cells ranging from the translocation of the chromosomes and vesicles to cell crawling and swimming - Wavelength ranges from around 1 um to around 1 mm  Electron microscopy, with its higher resolution, revealed that all eukaryotic cells are divided into similar multiple membrane limited compartments called organelles - Wavelength ranges from around 0.1 nm to around 100 um  Resolution ( resolving power) : the ability to differentiate between two very closely positioned objects  Resolution related numerically to D, minimum distance between two distinguishable objects; smaller the value of D, better the resolution  The resolution of the Light Microscope is about 0.2 um or 200 nm Bright-field Microscopy:  Shows live cultured cells  stained or unstained Phase-Contrast and Differential Interference Contrast (DIC) Microscopy Visualize Unstained living cells:  take advantage of the differences in the refractive index and thickness of cellular materials  produce image the differ in in appearance and reveal different features of cell architecture  both can be used in time-lapse microscopy in which the same cell is photographed at regular intervals to study cell movement Phase-contrast Microscopy:  cells are surrounded by alternating dark and light bands  degree of darkness or brightness of a region of the sample depends on the refractive index of that region  suitable for observing single cells or thin cell layers, but not thick tissues; also useful for examining the location and movement of larger organelles in live cells DIC:  cells appear in pseudorelief  based on interference between polarized light and is the method of choice for visualizing small details and thick objects  objects appear to cast a shadow to one side which primarily represents a difference in the refractive index  outlines of large organelles such as a nucleus or a vacuole can be seen  image is a thin optical section or slice through the object  details of can be observed in a species of such optical sections and the three dimensional structure of the object can be reconstructed by combining the individual DIC images Fluorescence Microscopy:  Can localize and quantify specific molecules in live cells  Allows for localization of proteins  Fluorescent: a chemical that absorbs light at one wavelength and emits light at a specific and longer wavelength  Light emitted by a fluorescent is used to form an image  Green-fluorescent protein (GFP) , a naturally fluorescent protein found in the jellyfish, can be introduced into specific cells of an animal; introduced protein will emit a green fluorescent when illuminated and thus, this could be used to localize the cells Immunofluorescence Microscopy:  Detects specific proteins in fixed cells  Antibody is covalently related to a fluorochrome  When a fluorochrome-antibody complex is added to a permeabilized cell or tissue the complex will bind to the corresponding antigen, which will then light up when illuminated by the exciting wavelength  Another variation : antibody is applied to the fixed tissue section, followed by a second fluorochrome-tagged antibody that binds to the common segment of the first antibody Confocal and Deconvolution Scanning Microscopy:  Enable visualization of three dimensional objects  Limitation to conventional microscopy: 1. Fluorescent light emitted by a sample comes from molecules above and below the plane of focus causing a blurred image 2. To visualize thick specimens, consecutive sections must be prepared, imaged, and
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