To provide a rapid, efficient and convenient method to detect one or more biological entities on a blotting membrane.

The detection can relate to the position, nature or amount of the biological substance on the membrane. The invention method involves a pressure assisted regiment for the supply and removal of reagents and permits washing of the contaminants from substances embedded in the membrane to be detected using very low volumes of liquid. This method enables completion of the blocking, washing and antibody binding steps in about 30 minutes without comprising blot quality. The device is comprised of several layers including a porous support layer below the blotting membrane, a flow distributor above the blotting membrane and a well on the flow distributor to contain the liquid to the desired area and to allow for lower starting volumes of such liquid. Preferably, the flow distributor is a non-binding or low binding hydrophilic porous membrane such as 0.22 micron membrane.