Western Blotting Flashcards
target proteins are transferred to a hydrophobic membrane after SDS-PAGE and detected using specific antibodies.
Western Blotting
important technique used in cell and molecular biology.
Western blotting
By using a western blot, researchers are able to identify specific proteins from
a complex mixture of proteins extracted from cells.
Western blotting technique uses three elements to accomplish this task
(1) separation by size,
(2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize.
After SDS Page, what happens?
a membrane is placed on the gel, to which the separated proteins in the gel are electrophoretically transferred to a membrane producing a band for each protein.
The membrane with transferred proteins is then
probed or incubated with a primary antibody (an antibody specific for the target protein). The unbound antibody is washed off leaving only the bound antibody to the protein of interest.
The bound antibodies are then detected by
developing the film which means reacted with a secondary antibody labeled with an enzyme, such as horseradish peroxidase (HRP). The bound enzyme activity is used to detect the target protein and visualized by a chemiluminescent or chromogenic method.
used to detect the target
protein and visualized by a chemiluminescent or chromogenic method.
bound enzyme activity
As the antibodies only bind to the protein of interest,
only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present.
Flow of western blotting procedure
analytical technique to separate proteins based on their molecular
weight.
SDS Page
When proteins are separated by electrophoresis through a gel matrix,
smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins.
In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely
eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.
detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that
cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length.
SDS
Polymerized acrylamide (polyacrylamide)
forms a mesh-like matrix suitable for the separation of proteins of typical size. The strength of the gel allows easy handling.
Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins based on their length in an easy, inexpensive, and relatively accurate manner.
