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Lecture 12

GENE 500 Lecture Notes - Lecture 12: Chromogenic, Chemiluminescence, ElectrophoresisPremium

2 pages15 viewsFall 2018

Course Code
GENE 500
Lawrence Grossman

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Genetics 500: Molecular Biology Introduction
LECTURE 12 - Immunoblots
Immunoblotting, also called Western blotting, combines the resolving power of gel
electrophoresis with the specificity of antibodies.
This multistep procedure is commonly used to separate proteins and then identify a
specific protein of interest.
Step 1: After a protein mixture has been electrophoresed through an SDS gel, the
separated bands (or spots, for two-dimensional gel electrophoresis) are transferred
(blotted) from the gel onto a porous membrane from which the protein is not readily
removed. Individual proteins (represented by blue ovals) are not visible at this stage
Step 2: The membrane is flooded with a solution of an antibody (Ab1) specific for the
protein of interest and allowed to incubate for a while. Ab1 binds to the protein of
interest (second from the top), but not to any other proteins attached to the
membrane, forming a layer of antibody molecules coincident with the protein (whose
position still cannot be seen at this point). Then the membrane is washed to remove
unbound Ab1
Step 3: The membrane is incubated with a second antibody (Ab2) that specifically
recognizes and binds to the first (Ab1). This second antibody is covalently linked to an
enzyme that catalyzes a chromogenic reaction or releases light (e.g.,
chemiluminescence), a radioactive isotope, or some other substance whose presence
can be detected with great sensitivity
Step 4: The location and amount of bound Ab2 are detected (e.g., by its color for a
chromogenic reaction or by detectors or film that measure the light released by
chemiluminescence), permitting the electrophoretic mobility (and therefore the mass)
of the protein of interest to be determined as well as its quantity (based on band
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