Figure. The binding activity of PRF1 with CRT.
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. Besides, Calreticulin (CRT) has been identified as an interactor of PRF1, thus a binding ELISA assay was conducted to detect the interaction of recombinant human PRF1 and recombinant human CRT. Briefly, PRF1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to CRT-coated microtiter wells and incubated for 2h at 37℃. 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 37℃. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of PRF1 and CRT was shown in Figure 1, and this effect was in a dose dependent manner.

Figure. Hemolysis activity of recombinant human PRF1.
The activity of recombinant PRF1 was measured by lysis of erythrocytes using a hemolysis assay. A general procedure is as fllows: two-fold dilute the recombinant human PRF1 with 0.9% NaCl, add 50μL a serial dilution of PRF1, 10μL 0.1M CaCl2 to each well, then add 50μL 0.25% rabbit erythrocyte (RaE) to each well and mixed gently. Add 10μL 0.9% NaCl to reaplace CaCl2 in control wells. The plate is incubated for 20 hours at 37℃, 5% CO2. The results are shown in Figure 2. It was obvious that the minimal effective concentration of PRF1 is 12.5μg/mL.
(A) 0.25% RaE tread with 12.5μg/mL PRF1 for 20h;
(B) Negative control (0.25% RaE tread with 12.5ug/mL PRF1) without CaCl2.