Lab Bradford Assay and SDSPAGE Western Blot Analysis How do

Lab: Bradford Assay and SDS-PAGE & Western Blot Analysis

How do antibodies interact with their target when examined at a molecular level?

Solution

One of the method to quantiy protein is Bradford assay .There is a non covalent interaction exist between coomassie and proteins , so it bind to the entire antibody and produce an antibody-coomssie dye complex.

SDS-PAGE is used to seperate the proteins. Treat the antibody with SDS and mercaptoethnol. The SDS molecule with negtive charge will coated with denatured Abs The denatured antibody fragments migrate through polyacrylamide gel depending upon the molecular weight (light hains and heavy chains) in an electric field.

In Western blot the interaction between antibody and antigen is highly specific The antibody can recognize either a protein or a peptide. The interaction is between an epitope, and the antigen binding siteon the antibody molecule, produse an antigen-antibody complex. Once the protein separated, it should transferred to nitrocellulose sheet. Then visualized dusing antibodies. First the primary antibody interct with the protein to form protein-antibody complex. Then by the addition of a secondary antibody, fc portion of the primary antibody will be recognised . The secondary antibody should be labeled with a compound. This will creates a coloured product when react with another compound.