- What can I do to induce starvation?
In NRK cells, starvation can be induced by changing the media to Hank's Balanced Salt Solution (serum-free) and incubating for 2 – 4 hours. Serum-free DMEM (Dulbecco's modified Eagle's medium) can be used, but the induction is weaker because DMEM contains amino acids.
- Tell me more about the inhibitors.
The well-known anti-malarial drug chloroquine has long been used as an inhibitor of lysosomal activity. Today, its efficacy as an anti-cancer drug is being studied. Bafilomycin A1 is a specific autophagy inhibitor used by many autophagy researchers.
Another commonly used inhibitor wortmannin (not included in this kit) blocks autophagy at an earlier stage.
- Two anti-LC3 antibodies are included. Are they used for different purposes?
Anti-LC3 antibody, clone 8E10 is conjugated to HRP and suitable for WB. For other applications, such as IC and IP, use clone 4E12.
- Can you tell me the details of the experimental protocol for LC3 detection by Western blotting?
Please refer to the Autophagy (LC3) FAQ.