in vivo sumoylation - (Mar/04/2009 )
Hi everybody,
I need some help regarding sumoylation. If anybody can help me with some suggestion on this topic since I am having a hard time these days trying to see in vivo sumoylation of my protein. it is already known that in vitro my protein is sumoylated but some how I can not see it in vivo (probably due to some technical problems, something is missing from my protocol). So, I express my protein (it is flag tagged) in cells, wash cells with cold 1xPBS, lysis of the cell in NP-40 buffer (supplemented with 25mM NEM), boil for 5 min in 2x Laemmli, centrifuge for 5 min on 13.000 followed by western blot using Flag antibody; my protein is there but I can not see the sumoylated form of it. I think something is missing from my protocol. Please help!!!
Any suggestions or information on protocol for in vivo sumoylation are more than welcome!
Thank you!
Since you are transiently transfecting your cells with your FLAG-tagged protein of interest, you likely have >600 fold over-expression of this protein. It is likely that the majority of your target will not undergo sumoylation due to limiting factors such as the level of available SUMO-1 and E2 conjugating enzyme/E3 ligases. Thus, you are likely missing the higher band due to a sensitivity difference between the two protein forms. You should look for sumoylation of the endogenous protein to avoid this problem. If sumoylation has already been shown, such evidence should have already been described. If this is not possible in your cell type, try staining for SUMO-1. Since sumoylation is covalent, you should see a band appear with a higher molecular weight than your protein of interest.
Hi Vedith,
The most common method of looking at sumoylation is through
overexpression of your target protein. This may not be a problem.
However most studies investigating Sumoylation co-transfect a Sumo expression vector as well.
This may be very important.
Some proteins are modified in vitro but not in vivo. Therefore, the most important step, in this and
every experiment you will do in your life, is .... use a positive control!
Go to the literature, find something that should be sumoylated in your system and use this as a positive control.
However I suspect the co-transfection with Sumo will work.
mikew on Mar 4 2009, 11:25 AM said:
The most common method of looking at sumoylation is through
overexpression of your target protein. This may not be a problem.
However most studies investigating Sumoylation co-transfect a Sumo expression vector as well.
This may be very important.
Some proteins are modified in vitro but not in vivo. Therefore, the most important step, in this and
every experiment you will do in your life, is .... use a positive control!
Go to the literature, find something that should be sumoylated in your system and use this as a positive control.
However I suspect the co-transfection with Sumo will work.
Hello Mikew,
Thank you for your reply.
In my message I didn't mentioned the fact that I tried to co-transfect my protein with Flag-SUMO and also I am using a positive control (p53 which it is well known to be sumoylated ), but I can not detect sumoylation not even on my positive control, this is the major problem. that's why I thought there might be a probleme with my technique.
Best wishes,
Edith
Hi again,
Sounds great!
In all the Sumo-p53 papers I've looked at
they co-transfect HA-Sumo.
That should fix your problem.
Good luck.