Western immunoblotting protocol; for Cleaved Caspase-3, Cleaved Caspase-9, Cleaved PARP, Caspase-9, and PARP
(as given by Cell Signaling Technologies)
previously...
SK MEL-28 human melanoma cells were seeded into 10 mL petri dishes at 200,000 cells per dish. After 48 hours, the cells were treated with 50 micromolar resveratrol. For information on resveratrol and these techniques, go to
and follow the links.
PROTOCOL:
1. The media was aspirated from the cells and the cells were washed 3 times with 5 mL PBS. The cells were scraped off and spun down to pellet at 1000Xg for 5 minutes.
2. Cell Extract Buffer was added to 5 volumes of the cell pellet. This lysed the cells. They were then resuspended in buffer , and frozen/thawed 3 times, which acted as a alternative to sonication. The pellet was spun down, and the supernatent was kept.
3. SDS Sample Buffer was added and the samples were heated to 95-100 degrees C for 5 minutes; they were then cooled on ice.
4. They were then microcentrifuged for 5 minutes, and 5 microliters of each sample was loaded into a 10cmX10cm SDS-PAGE gel.
5. 10 microliters of the untreated Cell Lysate Control was loaded in each lane.
6. The gel was then electrotransferred to a nitrocellulose membrane.
7. The nitorcellulose membrane was washed with 25 mL TBS for 5 minutes at room temperature; it was incubated in 25mL of Blocking Buffer for 1 hour at room temperature; and it was wshed 3 times for 5 minutes each with 15 mL TBST.
8. The membrane was incubated with primary antibody in 10 mL Primary Antibody Dilution Buffer with gentile agitation overnight at 4 degrees C.
9. The membrane was then washed 3 times for 5 minutes each with 15 mL TBST.
10. The membrane was then incubated with HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in 10 mL of Blocking Buffer with gentile agitation for 1 hour at room temperature; the membrane was then washed again as in step 9.
11. The membrane was incubated with 10 mL LumiGLO with gentile agitation for 1 minute at room temperature, drained of excess developing solution, wrapped in Saran Wrap, and exposed to x-ray film for 10 seconds.
