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Lecture 7

MBB 222 Lecture Notes - Lecture 7: Carboxymethyl Cellulose, Affinity Chromatography, Column Chromatography

Molec Biol & Biochem
Course Code
MBB 222
Edgar Young

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Fraction by solubility (centrifugation)
A tissue (like liver) is mechanically homogenized to break cells and disperse their
contents in an aqueous buffer. The density of the solution increases from top to bottom
that must be added to a density gradient (of a solute like sucrose with different
concentrations) to allow the large and small particles in suspension to be separated by
centrifugation at different speeds. Individual molecules sediment until their density
exactly matches that of the gradient so that each layer can be collected separately/
o A process in which cells are disrupted by homogenization that ruptures the
plasma membrane and releases the intact organelles
Extract / homogenate
o A solution where the proteins, after a cell membrane in ruptured, is released
into for protein purification procedure
o A process where centrifugal force is applied to the homogenate, containing the
proteins, causing them to separate into different fractions based on size or
charge and thus sediment at different rates
o Equipment driven by motor that spins the liquid samples at high speed
Centrifuge tube
o Tube filled with the solution to be centrifuged
Centrifugal force (g units)
o The radial force generated by a spinning rotor is expressed relative to the
earth's gravitational force
Pellet vs. supernatant
o Pellet: the solid precipitate formed at the bottom after centrifugation
o Supernatant: the remaining solution of the homogenate
Differential centrifugation
o Can be used to prepare subcellular fractions or to isolate specific organelles
Salting out (differential precipitation)
o Effect where at high salt concentrations the solubility of proteins is lowered
Ammonium sulfate
o Can be used due to its high solubility in water that at the right amount can
selectively precipitate some proteins while others remain in solution
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o Can be removed by dialysis
o A procedure that separates proteins from solvents by taking advantage of the
proteins’ larger size
Semi-permeable membrane
o Can be used to make a tube for the partially purified extract
o The membrane allows the exchange of salt and buffer but not proteins so they
remain within the membranous tube while the concentration of other solutes
in the protein preparation change until they come into equilibrium with the
larger volume of buffered solution outside the membrane
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Compare and contrast various chromatography modes used in protein purification ---
molecular property targeted by each technique; working principle, and deduce the order
of chromatographic separation of a series of proteins based on their molecular properties
(and vice versa), for various chromatography modes
Column chromatography
o Most efficient method of fractioning because it takes advantage of differences
in protein charge, size, binding affinity, and other properties
Stationary phase (beads / gel / resin / matrix) vs. mobile (aqueous) phase
o Stationary phase: a porous solid material with appropriate chemical properties
that is held in a column
o Mobile phase: a buffered solution that gradually filters through the porous
Gel-filtration (size exclusion / molecular exclusion) chromatography
o Separates proteins according to size
o Large proteins emerge from the column faster than small ones
o Larger proteins migrate through the column faster than smaller ones, because
they are too large to enter the pores in the beads and hence take a more direct
(shorter and faster) route around the beads
o The smaller proteins enter the pores and are slowed by the more complex path
Ion-exchange chromatography
o Uses differences in the affinity of protein due to the sign and magnitude of the
net electric charges (negative or positive) on the column affected by the pH
o The column matrix contains bound charged groups
Anionic: negatively charged ion
Cationic: positively charged ion
o Separation occurs as the pH and/or salt concentration (competing free salt ions
in the surrounding solution) of the mobile phase changes so as to create a pH
or salt gradient
Carboxymethyl (CM-) vs. diethylaminoethyl (DEAE-) cellulose matrix
o Used in ion-exchange chromatography
o Carboxymethyl cellulose matrix: has a carboxymethyl functional group that is
negatively charged (after ionization) at neutral pH making it a weak cation
o Diethylaminoethyl cellulose matrix: contains a diethylaminoethyl group that is
positively charged at neutral pH making it a weak anion exchanger
Affinity chromatography
o Based on the binding affinity of a protein
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