Question

how to use an antibody to probe or detect a protein.

Answer 1

To detect a protein, specific antibody against that protein is taken ( obtained by injecting the protein in suitable living animal and retreiving the antibody afterwards). It is called the primary antibody. After adding it, excess primary Ab washed out by using proper media/buffer solution. If there is no protein present, due to lack of binding site, all primary Ab will be out of the system. Then another antibody which is tagged with specific enzyme ( preferably which gives a prominent visual change during reaction with its substrate) and specific for that primary antibody ( antibody being a protein itself, another antibody against it from foreign and suitable living animal can be easily obtainable by following same procedure described above) is taken and added with the previously formed (if formed) protein-primary Ab complex. secondary Ab will bind with the primary Ab. Again after washing out the excess or all secondary Ab (in the scenario where no secondary Ab bound due to lack of protein-primary Ab complex) When the substrate of the enzyme will be added to this protein-primary-secondary Ab-Enz complex, It will give visual colour change (when protein is present) or remain unchanged (when potein is absent).

Radioactive isotopes can be used also in place of enzyme to tag with antibody where detection of radiation prove the presence of protein-Ab-secondary Ab complex denoting presence of the protein.