I treated my U87 MG astrocytes with TGF beta 100ng/ml in 12 well plate and infected with virus. I collected lysates and probed for Smad2 but I did not see any changes in phosphorylation. What could I do to troubleshoot my problem?
Hi Bibha,
I agree with Oliver's points. Yes, I would recommend a two hour serum starvation and then adding your TGFb in serum-free media. The growth factors within the serum very likely include smad2/3 agonists. Also, when collecting, I put my plate on a bed of ice and scrape the cells off in ice-cold RIPA buffer with phosphatase inhibitors, rather than dissociating from the plate, pelleting, and then lysing. In my hands, I've seen better p-SMAD signal (both 2/3 and 1/5/8) when I start lysis as quickly as possible, directly on the plate. Best of luck!
Thank you Sean. Could you also tell me how do you reconstitute your TGF beta? I reconstituted with 0.1% BSA in sterile water.
I have worked with activins and bmps, but not TGFb specifically. I would take a look at the spec sheet provided for your TGFb. some of these have required 0.1% BSA as a carrier while others may already contain BSA with the lyophilized protein. Also, some may require 4uM HCl.