proteasome inhibitor - wnt pathway (Apr/29/2011 )
I'm studying the Wnt pathway and would appreciate if someone could read my plan and check my calculations. I've got very limited time to finish my experiments and would appreciate all advice. Plus, my maths is not very strong.
I had checked LiCl effect on my cells and my data shows that overexpressing my gene of interest reduces b-catenin but my gene has no effect on b-catenin in the presence of LiCl. This demonstrates that it inhibits b-catenin via axin/gsk3/apc/b-catenin pathway. I now want to show if b-catenin is reduced via proteasome inhbition as a result of my gene of interest. I've decided to treat my cells with a proteasome inhibitor, MG132. I plan to transfect my cells with my gene and then treat the cells with proteasome inhbitor. Is my approach alright? Could you please double check my calculations:
MG132 from Sigma came in a vial with 5mg and it says it can be dissolved in DMSO. Since, I need to use uMol concentrations, I plan to add 1ml of DMSO to the 5mg MG132 powder to give me a stock solution of 10mM? The molecular weight of MG132 is 475.62.
Just ralised that making 2mM stock solution in DMSO would be better and have less error in dilution.
This means 2mg in 3ml of DMSO. The molecular weigjt of DMSO is 475.62.
Just read on the sigma website that If addition of a stock DMSO solution of MG132 to culture
medium results in formation of a precipitate, the following steps are recommended. Prepare the DMSO
stock solution at a concentration such that the final concentration of DMSO in the medium is approximately
0.2% DMSO (v:v).
I do not understand the advice by Sigma. If precipitate is formed, then what DMSO stock solution would have to be made to give me 5ml of 10uM, or 40uM of MG132 diluted in medium but the final DMSO in medium should not exceed 0.2% v/v?