Active Perforin 1 (PRF1)

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's plasma membrane, and oligomerises in a Ca2 dependent manner to form pores on the target cell. The pore formed allows for the passive diffusion of a family of pro-apoptotic proteases, known as the granzymes, into the target cell. The activity of recombinant PREF1 was measured by lysis of erythrocytes using a hemolysis assay. A general procedure is as fllows: two-fold dilute the recombinant mouse PREF1 with 0.9% NaCl, add 50μl a serial dilution of PREF1, 10μl 0.1M CaCl2 to each well, then add 50μl 0.25% rabbit erythrocyte (RaE) to each well and mixed gently. Add 50μl 0.9% NaCl to reaplace PREF1 in control wells. The plate is incubated for 20 hours at 37 ℃, 5% CO2. The results are shown in Figure 1. It was obvious that the minimal effective concentration of PREF1 is 2.5μg/ml.