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

20 views3 pages
8 Nov 2018
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
The three most widely used characteristics for
separating proteins are size, defined as either length or
mass; net electrical charge; and affinity for specific
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
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)
Unlock document

This preview shows page 1 of the document.
Unlock all 3 pages and 3 million more documents.

Already have an account? Log in

Get OneClass Notes+

Unlimited access to class notes and textbook notes.

YearlyBest Value
75% OFF
$8 USD/m
$30 USD/m
You will be charged $96 USD upfront and auto renewed at the end of each cycle. You may cancel anytime under Payment Settings. For more information, see our Terms and Privacy.
Payments are encrypted using 256-bit SSL. Powered by Stripe.