Western-Blotting

What process do immunoblotting or western blotting utilize to identify molecules in a myosin mixture?

They test potential antigens individually against antibodies.

How are potential antigens separated prior to western blotting?

They are separated by SDS gel electrophoresis.

What is the purpose of transferring proteins to a nylon or nitrocellulose membrane?

To keep the spatial distribution from the gel and make them accessible to antigen-specific probes.

What are primary antibodies used for in the western blotting process?

They are exposed to the membranes to specifically bind to the target antigens.

What type of secondary antibodies are used after primary antibodies in western blotting?

Enzyme-linked secondary antibodies.

What does the horseradish peroxidase enzyme do in western blotting?

It generates a detectable product that shows where the antigen is located on the membrane.

How are chromogenic and chemiluminescent detection different?

Chromogenic detection results in a colored precipitate visible to the naked eye, while chemiluminescent detection emits photons and requires sensitive instrumentation.

What is transblotting?

A technique for transferring proteins from the gel to the NC membrane using an electrical field that forces proteins to migrate laterally to the NC membrane.

What is the function of ponceau dye in western blotting?

It confirms that protein transfer occurred and allows correlation of membrane regions to gel lanes.

Why is Coomassie blue not used in western blotting?

It binds permanently to proteins and interferes with antibody binding.

What is a blocking agent and what is its purpose in western blotting?

A solution like 4% MP-PBS that blocks non-specific antibody binding.

What is the purpose of including Tween in PBST when washing the membrane?

To reduce non-specific binding between antibodies and proteins.

What is the difference between monoclonal and polyclonal antibodies?

Monoclonal antibodies recognize a single epitope, while polyclonal antibodies contain multiple different antibodies that bind to various epitopes.

Why should Tween 20 be avoided during the secondary antibody step?

Because it interferes with enzyme assays used in detection.

What is the function of peroxidase in western blotting?

It speeds up the reaction between (oxidizing) hydrogen peroxide and oxidizable substrate (4-chloro-1-naphthol).

How do you stop the reaction in western blotting after the enzyme reaction?

By transferring strips to deionized (DI) water.