Abstract: A rapid, efficient and convenient method to detect one or more biological entities on a blotting membrane is provided. The detection can relate to the position, nature or amount of the biological substance on one or more membranes. The invention method involves a pressure assisted regiment (such as vacuum or positive gas pressure) 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. In another aspect, the invention is directed to an apparatus useful in conducting the method of the invention.