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1. The membrane was clear after reaction with Phospho-stathmin, no other bands\stainingwere visible after a very long exposure. It was the first Ab I used with the membrane.
2. As mentioned, the cells were treated with a cocktail of protease and phosphatase inhibitor, however not with Calyculin A. I would appreciate if you could send me protocols orreferencesfor treating these cells.
3. Attached is a scan of one of the membranes after reaction with gERK.
Thanks a lot for your assistance.
Have a nice week.
Asked on Aug 02 2012
Thank you for your reply and for kindly providing this information.
Reviewing the details, I can suggest my main concern is still that the cells have not been treated.
Here is the treatment which was used in testing the cells (as shown in the WB image on the datasheet, HeLa):
1. The cells were starved for 24hr in the medium without FBS.
2. Treated the cells with 20% FBS for 3h. 3.
Added calyculin A at the concentration of 100ng/mln.
4. After 30min, the cells were harvested and lysed.
I hope this will be helpful. Please do not hesitate to let me know the results. I hope we can resolve this case as soon as possible.
Answered on Aug 02 2012