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Thanks a lot for your suggestions. The customer would like to try this protocol this week. Do you have information regarding thelysis buffer? Does she need another phosphatase inhibitor or the cocktail she used should be fine?
100ng/ml calyculin for 30min in 37degrees, right?
Thank you in advance for the additional info.
Have a nice week.
Asked on Aug 06 2012
Thank you for your message.
1. Growing cells were starved for 24hr in the medium without FBS.
2. Growing cells treated the with 20% FBS for 3hr.
3. Add calyculin A to the growing cells at the concentration of 100ng/mln for 30 minutes at 37oC.
4. After the 30 min incubation, the cells were harvested and lysed with RIPA buffer containing the usual protease inhibitor cocktail.
I hope this will be helpful. Should you have any further questions, please do not hesitate to contact us.
Answered on Aug 06 2012