Testing Specificity of Commercially Available Nicastrin Antibodies

A protein complex known as γ‐secretase is involved in the development of Alzheimer's disease, which affects cognitive functions such as learning and memory. Nicastrin, a component of γ‐secretase, is the substrate receptor in the protein complex. In a previous research project, the expression of the nicastrin protein was tested by immunofluorescence in a line of nicastrin knockout cells. However, the antibodies stained structures in both wild‐type and knockout cells. This result suggested either of two possibilities: the cells were not nicastrin knockout cells, or the antibodies were nonspecific to nicastrin. Genotyping by polymerase chain reaction (PCR) determined that the nicastrin gene itself was, in fact, knocked out of the cells. To determine if the nicastrin antibodies are specific, western blots were run using the same antibodies with lysates from both wild‐type and knockout cells. By revealing cross‐reactive bands in both lysates, western blots will also aid in determining why the antibodies bind to both knockout and wild‐type cells in immunofluorescence microscopy. Therefore, determining the specificity of commercially available antibodies will better inform both ourselves and the scientific community about using these antibodies for experiments. Support or Funding Information Funding contributed by the International Healthspan Institute.