Mouse Perforin protein (active)

Perforin 1 (PRF1) is a pore forming cytolytic protein found in the granules of cytotoxic T lymphocytes (CTLs) and NK cells. Upon degranulation, perforin binds to the target cell'Splasma membrane, and oligomerises in a Ca2 dependent manner to form pores on the target cell. Perforin is thought to act by creating holes in the plasma membrane which triggers an influx of calcium and initiates membrane repair mechanisms. These repair mechanisms bring perforin and granzymes into early endosomes. Besides, Granzyme A (GZMA) has been identified as an interactor of PRF1, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse PRF1 and recombinant mouse GZMA. Briefly, PRF1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to GZMA-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-PRF1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of PRF1 and GZMA was in a dose dependent manner.