[shininglight]

hi, could someone explain how after a western blot is treated with substrate it is developed in dark room with x ray film and where the antibody attached to protein on the membrane a black spot appears , i know the procedure i just dont understand how it happens, thanks

[sdekivit]

after transferring the proteins to the sheet antibodies (primary antibody) is incubated that specifically binds to the protein pof interest.

next a second antibody directed against the constant region of the primary antibody (say the first antibody is derived from a mouse, the second antibody derived from a goat is directed to mouse antibodies)

--> the second antibody is conjugated with a radioactive label that can be made visible on an autoradiogram (x-ray film)