General principle of the PK-PET blot technique. Paraffin-embedded paraffin sections are blotted onto nitrocellulose membrane and dried. After deparaffinization and rehydration, the diagnostic blots are reacted harshly with the proteolytic enzyme PK (right), while control blots are incubated without PK (left). The enzymatic reaction is then quenched and followed by immunodetection of α-synuclein using an appropriate primary antibody. Immunoreactive profiles are visualized by binding of a matched secondary antibody coupled to alkaline phosphatase (AP) which reacts with a colorigenic formazan dye. While undigested PET blots display the abundantly expressed "normal" α-synuclein (lower left), the PK-PET blots reveal the pathological protease-resistant α-synuclein (lower right). This procedure provides the basis of a novel diagnostic method to detect and characterize protein deposits in neurodegenerative diseases. Figure courtesy of Manuela Neumann and Philip Kahle.