Lab 9 - Western Blotting Flashcards
What are the tasks of this lab?
Membrane blocking step, primary antibody incubation, secondary antibody incubation, chemiluminescence reaction and visualization
What is immunoblotting (Western Blotting) used for?
To detect macromolecular antigens (proteins) that react with specific polyclonal or monoclonal antibody by using the resolving power of SDS-polyacrylamide gel electrophoresis with a highly sensitive antibody binding assay to detect a specific protein in a mixture of different proteins
What happens in the SDS-polyacrylamide gel?
Proteins are first separated
What happens in the electroblotting step?
Proteins are transferred to a sheet of nitrocellulose of PVDF membrane
How do the proteins stick to the membrane?
In the same position that they had in the SDS-polyacrylamide gel
What blocking protein can be utilized in the next step?
Serum albumin or dry milk
What is a blocking protein used for?
To coat the areas of the membrane which do not contain any proteins
What is done to the primary antibody?
It is chosen to bind only to the protein of interest and is then incubated with the membrane blot
What is done after a washing step?
A secondary antibody, which binds specifically to the conserved protein of the primary antibody is incubated with the membrane
What is the secondary antibody conjugated to?
An enzyme (horseradish peroxidase)
What do the conjugate enzymes do?
Catalyze a chemiluminescent reaction that allows visualization of as little as 1 pg of protein
How do we visually detect the presence of our protein of interest?
Utilize a non-radioactive light emitting procedure that is designed to detect proteins that are immobilized on a membrane
What does the chemiluminescence reaction involve?
The emission of light by oxidation of luminol by an enzyme, horseradish peroxidase, that is conjugated to the secondary antibody. Following the addition of the chemiluminescence reagents to the membrane, oxidative degradation of luminol occurs, resulting in light emission at a wavelength of 428 nm (light emission is short-lived and can be captured by gel imaging system).
What are the required lab results?
ECL and light electronic images of your membrane
- Chemiluminescence blot
- Coulometric blot
