I would try a 15% gel since the protein is quite small. You also might want to consider doing a semi-dry blot (if you are not already doing this). In order to be of better help to you, I would like to have a look at your protocol.

Caspase 3 is a pain for everyone. Try the 15% gel but you may need to increase the amount of protein you are loading. In a 10 well minigel from brain I had to run at least 50ug protein. The semidry blot can also help.

Also you may need to try a number of different antibodies.

Good luck. you will need it

thanks ever so much 

Hi Syed,

Is you antibody compatible/tested on hamster lysates? Are you struggling with general caspase 3 detection or cleaved/activated caspase 3? I would suggest that you have a positive control like influenza infected cells (please see https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection

OR http://www.sciencedirect.com/science/article/pii/S0042682214000117) just to help you pin point where you could optimize you assay. You can use cycloheximide/sts or cpt (please see https://www.researchgate.net/publication/51069147_Flavivirus_NS4A-induced_Autophagy_Protects_Cells_against_Death_and_Enhances_Virus_Replication for possible concentrations). In the first paper I attached we used anti- active/cleaved caspase 3, which works well when studying programmed cell death. Best of luck!

-ED

Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...

Article Flavivirus NS4A-induced Autophagy Protects Cells against Dea...

thanks ever so much

Dear Anouk A J Hamers, Syed Zahid Ali Shah, and Sarah Zilka How long the time for transferring the protein from the gel into PVDF membrane using semidry transfer? I am also dealing with this situation. Thank you in advance