The production of beta galactosidase can be stimulated by adding lactose or the lactose analog, IPTG to the bacterial growth medium. To study the time course of beta galactosidase production chloramphenicol, which prevents m-RNA from attaching to the ribosome, can be added at 15 minute intervals. Since IPTG activates the DNA-protein pathway, the amount of beta galactosidase produced should be related to how long the pathway is active before chloramphenicol is added.
Once produced, the beta galactosidase can be isolated from the bacteria along with other proteins. It is then seperated from other proteins using SDS PAGE Gels coupled with Western Blotting.
Separating beta galactosidase from other proteins in the cell involves two electrophoretic steps, SDS-PAGE gels and Western Blotting . SDS-PAGE gels separate proteins on a gel, but it can be difficult to detect minor ones. Once proteins are seperated by SDS-PAGE, they can be transfered to a Western Blot, which makes use of antibodies to detect specific proteins.
SDS PAGE Gels
SDS - (sodium dodecyl sulfate) is a detergent that helps disrupt cell membranes. You used it last week to help disrupt the cell membrane of the E. coli. It also linearizes proteins by disrupting their three dimensional structure and coats them with negative charges. This means they will migrate through a gel based on their molecular weight rather than their structure.
PAGE - (PolyAcrylamide Gel Electrophoresis) - is the use of a gelatinous matrix made of polyacrylamide and an electrical current to separate proteins.
This week you will use SDS PAGE gels to isolate the beta-galactosidase produced by E. coli. The beta galactosidase produced by the E. coli was collected after protein synthesis was stopped by adding chloramphenicol to the cultures. The cultures were then spun to pellet the cells and the supernatant was removed and the pellet was resuspended in SDS-PAGE loading buffer. The cultures were then stored at -80oC.
Major steps in running SDS PAGE gels
Antibodies used in the Western Blot
The proteins that were separated by SDS PAGE will now be further isolated and moved onto a membrane using an electrical current. The beta-galactosidase will then be detected by using two different antibodies.
The primary antibody is derived from a rabbit and will bind to the target protein, beta-galactosidase.
Thesecondary antibody is derived from a goat and will bind to any antibody produced by a rabbit. For this reason, it will bind to the primary antibody.
Link the goat anti-rabbitt antibodies to alkaline phosphatase.
Click to see the relationship between the primary and secondary antibodies and beta-galactosidase.
Sequence of Events in a Western Blot
Blotting moves the proteins from the SDS PAGE gel onto a nitrocellulose membrane.
After the proteins are transferred to the nitrocellulose membrane, it can be stained with Ponceau Red stain. This is done to confirm proteins have been transfered from the gel to the membrane.
Blocking is done by incubating the membrane in dry milk blocking solution. This floods the membrane with proteins that will bind randomly across its entire surface. The presence of these proteins helps assure specific binding by the primary and secondary antibodies.
Probe the membrane with a solution containing the primary antibody. This is the anti-beta-galactosidase antibody derived from rabbits.
Wash the blot with detergent buffer to remove excess antibody
Note the specifically bound primary antibody.
Probe membrane with a solution containing the secondary antibody. This is anti-rabbit antibody derived from goats. It is coupled with alkaline phosphatase enzyme.
Wash the blot with detergent buffer to remove excess antibody.
Transfer the blot into a developing buffer that will cause a blue precipitate to form where the secondary antibody is located on the gel.
Photograph and analyze the results
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