GENE 500 Lecture Notes - Lecture 11: Sodium Chloride, Two-Dimensional Gel Electrophoresis, Polyacrylamide Gel Electrophoresis

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8 Nov 2018
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Lecture 11
Purifying, Detecting & Characterizing Proteins
A protein must be purified to determine its
structure and mechanism of action
Molecules, including proteins, can be separated
from other molecules based on differences in physical
and chemical properties
Centrifugation
Electrophoresis
Chromatography
The three most widely used characteristics for
separating proteins are size, defined as either length or
mass; net electrical charge; and affinity for specific
ligands
Centrifugation
The principle behind centrifugation is that two types of
particles in suspension (cells, cell fragments,
organelles, or molecules) with different masses or
densities will settle to the bottom of a test tube at
different rates
Chromatography
A) Gel filtration chromatography separates proteins that differ in size. A mixture of
proteins is carefully placed, or loaded, on the top of a cylinder packed with porous
beads. Smaller proteins travel through the column more slowly than larger proteins.
Thus the different proteins emerging in the eluate flowing out of the bottom of the
column at different times (different elution volumes) can be collected in separate tubes,
called fractions.
B) Ion-exchange chromatography separates proteins that differ in net charge in columns
packed with beads that carry either a positive charge (shown here) or a negative charge.
Proteins having the same net charge as the beads are repelled and flow through the
column, whereas proteins having the opposite charge bind to the beads more or less
tightly, depending on their structures. Bound proteinsin this case, negatively charged
proteinsare subsequently eluted by passing a salt gradient (usually of NaCl or KCl)
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