Western-Blotting

Immunoblotting or western blotting answers the question of what molecules are present within the myosin mixture by testing potential antigens individually against antibodies

Potential antigens are first separated by SDS gel electrophoresis, then transferred to a sheet of nylon or nitrocellulose membrane (keeps the spatial distribution from the gel)

Removing the proteins from the gel and exposing them on the membrane makes them accessible to antigen-specific probes

The membranes are first exposed to specific primary antibodies, after the primary antibodies are probed using enzyme-linked secondary antibodies. The enzyme (horseradish peroxidase or alkaline phosphatase) generates a detectable product that shows where the antigen is located on the membrane.

Chromogenic detection -> the product is a colored precipitate visible to the naked eye

Chemiluminescent detection -> the product is emitted photons which are detected with sensitive instrumentation

More sensitive than chromogenic detection

Transblotting –> technique for transferring proteins from the gel to the NC; involves applying an electrical field to force the proteins to migrate laterally to the NC membrane

Staining the membrane with ponceau dye confirms that the transfer of proteins from the gel to the NC membrane has occurred and allows for correlating specific regions of the membrane to specific lanes in the gel

Coomassie blue is not utilized because it binds permanently to the proteins and interferes with the binding of the antibodies

Ponceau dye disassociates with the proteins during wash steps

Blocking agent (western) - > 1.6g dehydrate milk powder to 40 mL of PBS (not PBS-T) makes 4% MP-PBS

Milk powder can contaminate, needs to be fresh for each use

Blocks non-specific antibody binding

Best to block the membrane while it is intact

After initial blocking, decant and replace with PBST (Tween included to reduce non-specific binding between antibodies and proteins other than their antigens)

Antibody buffer (western) -> 0.5% MP-PBS; 0.1g dehydrated milk powder in 20 mL PBS

Monoclonal antibodies -> ones that recognize just a single epitope out of the many that are present within an antigen

Polyclonal antibodies -> (like the mouse serum) contain many different antibodies which bind to all epitopes present within an antigen

During secondary antibody step rinse without tween 20 because it interferes with enzyme assays used in detection

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