Hi,
I am planning to treat my cells (transiently transfected and I generally process transfected cells after 48 hours of transfection) with MG132 for preventing degradation.
From literature, 10 um MG132 for 4 hours sounds safe but I am not sure exactly when (specifically how many hours after transfection) it would be ideal to treat the cells in culture with MG132?
Should I add it 4 hours before lysis?
Any advice would be really helpful!
Thanks.
Hi Tanmoyita
Depends what you are trying to understand. Once it gets into the cells, MG132 will block the proteasome (as well as other proteases...) almost instantly . What happens next is really system-dependent. Proteasome-processed material will start to accumulate (Ub-XYZ) over time, stress responses will kick in etc... Would suggest you try a dose curve to look at your favorite endpoint measurement...most likely 100 nM should be enough if the proteasome is involved; I think the historic 10 uM is based on lot of magic dusting and the idea that some cells may not take it up so easily, so brute force feels safer. Regardless, long term treatments (subjective but let's say 16 h+), may cause so much stress your cells will start to die on you (may not be obvious yet....but they will down the road). So yes, 1 to 6 h is a good way to start (just time-course it).
Good luck!
S
S
Hi Tanmoyita
Depends what you are trying to understand. Once it gets into the cells, MG132 will block the proteasome (as well as other proteases...) almost instantly . What happens next is really system-dependent. Proteasome-processed material will start to accumulate (Ub-XYZ) over time, stress responses will kick in etc... Would suggest you try a dose curve to look at your favorite endpoint measurement...most likely 100 nM should be enough if the proteasome is involved; I think the historic 10 uM is based on lot of magic dusting and the idea that some cells may not take it up so easily, so brute force feels safer. Regardless, long term treatments (subjective but let's say 16 h+), may cause so much stress your cells will start to die on you (may not be obvious yet....but they will down the road). So yes, 1 to 6 h is a good way to start (just time-course it).
Good luck!
S
S
Hi Tanmoyita,
I would like to add that it is very subjective and might vary from cell to cell. I once started to work with MG132. I faced problem while treating different cell types, with different point of sensitivity. Please make sure you do a triplicate analysis of the same, in a dose dependent manner and choose your dose. Also, try to see with the chosen dose, a time dependent point so that you come to a second dimensional conclusion as far as the dose and time is concerned! You may put the points of your study in the supplement, which will increase the value of your manuscript.
Best,
Somnath.
Thank you Somnath da!
I thought the same and planned to try different doses as you mentioned!.
But can you clarify one thing.. how stable this MG132 is inside the cells?
Hi, can I use MG132 on fission yeast? If yes, how long I need to treat the cells?
Hello everyone, I am working on a project for hiv 1 integrase. I have performed cloning and according my seq results it was successful. It has been few weeks I’m trying to detect my protein on western but it doesn’t show up. Hiv 1 integrase is getting degraded so I am using proteasome inhibitor. I have tried several treatments but none of them has worked. I did 50ul for 3 and 4 hours, 20ul for 4 hours, 10ul for 8hours and 6hours, 2ul for 18hours. I seed the cells and thennafter 24 hours I make the transfection with my pcmv5 flag integrase plasmid and then after 24 hours I start the treatments. Cell line used is Hek cellsZ any advice is valuable. Thank you
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