Adipocytes ChIP - Woes on sonicating adipocytes (Jun/26/2008 )
We are trying to sonicate adipocytes (3T3-L1)for ChIP, but the fats ( or we susepct) the fats are making things very difficult for us, we could not get the DNA to our desired range, we did not have this problems for liver cells.
Also, it's common for ppl to use SDS during sonication. We tried that but it not good enough (it did show some improvement). Actually just as a very general Q, would SDS denature the protein, which would pose problems when we do IP (I know we usu dilute the SDS x10 for IP, but still there is SDS)
I think our main problem is that we need to get rid of the fats before sonication, and we dont know what is the best way to do that.
Thank you!
-chipgal-
QUOTE (chipgal @ Jun 26 2008, 08:56 PM)
We are trying to sonicate adipocytes (3T3-L1)for ChIP, but the fats ( or we susepct) the fats are making things very difficult for us, we could not get the DNA to our desired range, we did not have this problems for liver cells.
Also, it's common for ppl to use SDS during sonication. We tried that but it not good enough (it did show some improvement). Actually just as a very general Q, would SDS denature the protein, which would pose problems when we do IP (I know we usu dilute the SDS x10 for IP, but still there is SDS)
I think our main problem is that we need to get rid of the fats before sonication, and we dont know what is the best way to do that.
Thank you!
Also, it's common for ppl to use SDS during sonication. We tried that but it not good enough (it did show some improvement). Actually just as a very general Q, would SDS denature the protein, which would pose problems when we do IP (I know we usu dilute the SDS x10 for IP, but still there is SDS)
I think our main problem is that we need to get rid of the fats before sonication, and we dont know what is the best way to do that.
Thank you!
When you centrifuge the cells after lysis (but before sonicating) do you get a pelicle of lipids floating on top. If so, you could always try removing them directly instead of solublizing. Also, it may not be the problem of the lipids since there are many reasons why different cell types have diffent efficiencies with sonication. You might just try reducing the concentration of cells used during sonication or just sonicate more. In any case, I'll be interested to know if you figure it out, since I'll be trying ChIP on adipose tissue soon.
-KPDE-
QUOTE (KPDE @ Jun 27 2008, 11:34 AM)
QUOTE (chipgal @ Jun 26 2008, 08:56 PM)
We are trying to sonicate adipocytes (3T3-L1)for ChIP, but the fats ( or we susepct) the fats are making things very difficult for us, we could not get the DNA to our desired range, we did not have this problems for liver cells.
Also, it's common for ppl to use SDS during sonication. We tried that but it not good enough (it did show some improvement). Actually just as a very general Q, would SDS denature the protein, which would pose problems when we do IP (I know we usu dilute the SDS x10 for IP, but still there is SDS)
I think our main problem is that we need to get rid of the fats before sonication, and we dont know what is the best way to do that.
Thank you!
Also, it's common for ppl to use SDS during sonication. We tried that but it not good enough (it did show some improvement). Actually just as a very general Q, would SDS denature the protein, which would pose problems when we do IP (I know we usu dilute the SDS x10 for IP, but still there is SDS)
I think our main problem is that we need to get rid of the fats before sonication, and we dont know what is the best way to do that.
Thank you!
When you centrifuge the cells after lysis (but before sonicating) do you get a pelicle of lipids floating on top. If so, you could always try removing them directly instead of solublizing. Also, it may not be the problem of the lipids since there are many reasons why different cell types have diffent efficiencies with sonication. You might just try reducing the concentration of cells used during sonication or just sonicate more. In any case, I'll be interested to know if you figure it out, since I'll be trying ChIP on adipose tissue soon.
Thanks! i am now doing more washes for my cells when I lysed them. Hope that it work! have a good weekend!
-chipgal-