The selectivity of the inhibitory component of GTB has previously been studied by comparing IC 50 values for csecretase and other proteases, showing a,60-fold difference compared to the aspartyl protease HIV-1, and even higher differences compared to other types of proteases. In the GTBproximity ligation method developed here, the specificity is increased, due to the fact that proximity ligation requires two detection probes. One detection probe was directed to GTB and the other to primary antibodies to PS or nicastrin. Our novel method thus secures the specificity of GTB for c-secretase to a level beyond the specificity of the inhibitor component on its own. Another interesting observation was that there were relatively fewer PLA signals in the nuclear/perinuclear region in the PLA assay using the nicastrin antibody and GTB than in the assay using the nicastrin and PS antibodies. Both the finding that the interactions between active c-secretase and nicastrin constitute only a portion of the total amount of PS1-nicastrin interactions, and that the PLA signals obtained by the GTB assay were located more peripherally compared to the PLA assay using only antibodies, are in line with the notion that c-secretase needs to mature to become active. It has not, however, been established where the maturation occurs. Our data with the GTB method thus gives novel information as to where c-secretase matures, which appears to be distal from the perinuclear ER system. In conclusion, we present a novel proximity ligation-based method to visualize active c-secretase. By comparing the results from this method with traditional PLA in neurons, we enable differentiation of active from inactive c-secretase in the cells. The technique is Lapatinib distributor highly sensitive and allows detection of single complexes. Since the assay is conducted in situ, it allows determination of the subcellular location of active enzymes as well as their association with other proteins. The selectivity of the assay for active enzymes is achieved by using an active site inhibitor of c-secretase, which is conjugated to a PEG linker, a photoreactive group and a biotin group. The assay has dual specificity because it requires both an active site and an additional c-secretase component to give a signal. With all these advantages over more traditional Everolimus mTOR inhibitor techniques for studying membrane protein interactions, this method opens the doors for a multitude of applications. For instance, by combining the GTB assay with subcellular marker staining and high resolution microscopy, such as STED, a detailed analysis of the subcellular location of the active enzyme complexes could be done.