Dissociated cells were washed with phosphate-buffered saline (PBS) two times, resuspended in 500 μL binding buffer, and then incubated for 1 hour at room temperature in the presence of 0.5 μg/mL annexin V-FITC (R&D) and 5 μL propidium iodide for 5 minutes in bindingbuffer as described by the manufacturer.
The pellet was then re-suspended in 200 μl binding buffer and then centrifuged again at 1200 rpm for 5 min and the supernatant removed.
Cells were resuspended in 195 µL of binding buffer, and then incubated with annexin V-FITC and PI for 15 min in the dark at room temperature.
All suspensions were washed three times with PBS, suspended in 500 μl of binding buffer, and then analyzed by flow cytometry (Epics XL, Beckman Coulter, USA) using FLOWJO 7.6 software.
Cells were rinsed with 1× binding buffer and then incubated with 200 μl of 1× binding buffer containing 5 μl of the annexin V solution and 10 μl of the PI solution; then, 5 μg/ml of Hoechst 33342 (H1399 from Invitrogen) was used for nucleus staining (blue).
The protein-bound resin was loaded onto a column, washed with 20 ml of binding buffer and then washed sequentially using the binding buffer containing 20 and 40 mM imidazole.
Cells were washed with FACS buffer and resuspended in 100 µl of annexin V binding buffer and then FITC-annexin V was added (5 µg) to each well according to the manufacturer's protocol (ApoDETECT FITC-Annexin V Kit; Zymed Laboratories, San Francisco, CA, USA).
For pulldowns by His6-Prp19(144–503), ∼1 µg yeast protein lysate was mixed with 50 µl (1∶1) slurry of beads that were incubated first for 1 hr at 4°C with a solution of 1 µg/ml BSA, washed once with NP-40 buffer with 5 mM imidazole, twice with bead binding buffer and then either beads alone or with recombinant fusion protein.
The cells were suspended in binding buffer and then analyzed by flow cytometry.
After removing unbound I-echistatin, thydrophiliclic PVDF filters were washed 3× with the binding buffer, and then collected.
