A protoype to isolate differentially expressed proteins (Pds) by protein subtraction was presented. Two sets of protein mixture A and (A-Pd) were prepared. The mixture A contained bovine serum albumin, ovalbumin, a-lactalbumin, trypsinogen and carbonic anhydrase while the mixture (A-Pd) was the same as the mixture A except that one of the proteins acting as a differentially expressed proteins was lacking. Rabbit antisera were raised against the mixture A. Proteins in the mixture (A-Pd) were then coupled to CNBr- activated Sepharose 4B in order to prepare the affinity column. The antisera were applied to the affinity column and the pass-through exclusively contained the antibodies that specifical1y reacted with the differentially expressed proteins. After three cycles of successive affinity chromatography, the differentially expressed proteins from the mixture A was isolated following by immunoprecipitation. The SDS-PAGE analysis demonstrated that the differentially expressed proteins was effectively isolated by this method of