Can we co-incubate the primary and secondary antibodies at the same time for Western Blotting ?

Primary and secondary antibodies should not be incubated at the same time. Incubating primary and secondary antibodies together has the potential to form a large complex of antibodies which never binds to the antigen on the membrane. Primary and secondary antibodies also require different incubation times. In addition, not applying the primary and secondary antibodies sequentially also makes it more difficult to analyze poor results, since it would hard to discern whether the result is due to the primary, the secondary, or their interaction.