Blot membranes blocking

Period 2 Membrane blocking and incubation of blot with Con A-HRP and substrate solution. 

B 3. An economical way to block a blotted membrane is to incubate it in a 10% solution of nonfat milk powder. How does this solution function as a blocking reagent ... 

Place the blot in blocking buffer (> 1 mL/cm2 membrane), and incubate on a rocking table for 1 h at room temperature or overnight at4°C, whichever is more convenient... 

Incubate the blot(s) in membrane blocking solution for 30 min, allowing 0.25 mL/cm membrane see Note 7). 

After transfer or staining, submerge the membrane blot in blocking buffer in a container. 

Figure 8.11 Specific enzymatic immunodetection of a blotted protein. Depicted are blocked binding sites on the membrane (1), a primary antibody (2) specifically bound to an antigenic protein, and a secondary antibody (3) bound to the primary antibody. The secondary antibody is conjugated to a reporter enzyme (4). Substrate (S) is converted to insoluble product (P) at the site of the antigen.

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

Once the transfer is completed, the blotting system is carefully disassembled. The filter paper and gel are removed and the membrane is placed in TBS buffer for 10 min. Finally, the membrane is soaked in blocking buffer for 60 min. 

Proteins are blotted onto a nitrocellulose membrane by diffusion for 1 h at 60°C. Non-specific sites on the nitrocellulose membrane are blocked with 50 g/1 powdered milk dissolved in PBS/0.1% Tween-20. The membrane is washed in PBS/0.1%... 

Blots can be reversibly stained with Ponceau S to confirm transfer has taken place This must be done before the blocking stage, since after this, the membrane is saturated with protein This is not a highly sensitive stain—only major bands are revealed... 

Fluorescamine, or 4-phenylspiro[furan-2(3//),l/-phthalan]-3,3,-dione, is used to introduce a fluorescent label on electroblotted proteins via reaction with free amines. Transferred proteins are visualized on blot transfer membranes with UV light. This stain can be very sensitive and can be used in conjunction with a second detection method such as immunoblotting (also see Basic Protocol 3). However, the protein is irreversibly modified because fluorescamine reacts with available amino groups (i.e., lysines and the protein N terminus if it was not previously blocked). 

---