My secondary antibody for western blot is placed in the "amber" vial to protect from the light. However, while making dilution of the secondary antibody it is exposed to light for 2-3 minutes. Does it really affect the quality of the secondary antibody, especially in having unspecific binding?
Chandra K Dixit Thanks. I am just wondering why there's some unspecific bindings I got in my blot? Though, my protein band is well enough, but still there's some unspecific bindings have been found. If the secondary antibody is old, it may give unspecific bindings?
You may try using fresh primary and secondary antibody along with fresh PBST for wash. Wash the membranes in 1x Pbst for 15 min /4 time after primary and secondary antibody incubation..
Light won't affect ur HRP secondary antibody.
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