Question

in the process of immunoblotting why is it important to block the membrane after the transfer step? also further explain why we beed to do washings after the incubation with primary and secondary antibodies?

plz thoroughly

Answer 1

Blocking is done to block all the unreacted site present on membrane in order to reduce the amount of non specific protein binding in further step. Most commonly used blocking agents are BSA, non fat dry milk, gelatin, casein. Buffer used for blocking person are TBS/PBS. If blocking is not done then added antibody can binds at non specific positions on membrane that will give false positive results.

After incubation of primary or secondary antibodies washing is done to remove the unbound antibodies from membrane because all of the antibodies are not bound with antigen present on membrane. If washing is not done, non specific bands appears on membrane due to presence if unbound antibodies.