What is the best nuclear protein marker? - Fractionation (Dec/04/2005 )

Hi all,

I have been working on cytoplasmic/nuclear fractionation using several mammalian cells.
I used Ku86 as nuclear marker, but I recently found out that this protein is regulated by DNA damage. Since I'm studying the regulation of p53 upon DNA damage, I guess Ku is not suitable as the marker for me anymore. Does anyone know a good nuclear protein marker? Is LaminA/C a good candidate?

Another problem I run into was that nuclear proteins always leaked out into the cytoplasmic fraction. I used hypoionic condition (10mM KCl, 0.5mM MgCl2, 20% glycerol and 0.1% NP40). Should I leave out the detergent? Or if the solution density or the spin down condition (1000g) was not enough for precipitating all nucleus?

I also found that the fractionation condition I used was ok for WS1 (fibroblast cells), but for MCF7 (epitheial cells) the fractionation was completly messed up!! Any suggestion on how to modify the protocol?

I'm sorry for so mamny questions. Thanks for help in advance!!

Yuanita

Hi
You can use Ab against histone proteins in the nucleus. As far as the lysis is concerned make sure that the nuclear pellet is once rinsed with ice cold PBS once (favourably with phosphatase/protease inhibitors) so that there is no carry over proteins of the cytoplasmic fraction
Ope this helps

Hi all,

I have been working on cytoplasmic/nuclear fractionation using several mammalian cells.
I used Ku86 as nuclear marker, but I recently found out that this protein is regulated by DNA damage. Since I'm studying the regulation of p53 upon DNA damage, I guess Ku is not suitable as the marker for me anymore. Does anyone know a good nuclear protein marker? Is LaminA/C a good candidate?

Another problem I run into was that nuclear proteins always leaked out into the cytoplasmic fraction. I used hypoionic condition (10mM KCl, 0.5mM MgCl2, 20% glycerol and 0.1% NP40). Should I leave out the detergent? Or if the solution density or the spin down condition (1000g) was not enough for precipitating all nucleus?

I also found that the fractionation condition I used was ok for WS1 (fibroblast cells), but for MCF7 (epitheial cells) the fractionation was completly messed up!! Any suggestion on how to modify the protocol?

I'm sorry for so mamny questions. Thanks for help in advance!!

Yuanita

[quote name='rajgene' date='Dec 6 2005, 07:44 AM' post='33618']
Hi
You can use Ab against histone proteins in the nucleus. As far as the lysis is concerned make sure that the nuclear pellet is once rinsed with ice cold PBS once (favourably with phosphatase/protease inhibitors) so that there is no carry over proteins of the cytoplasmic fraction
Ope this helps

Thanks for the info, Rajgene.
Actually, using histone as marker might give some false results because histones are always in the pellet after separation. I always can detect histone H3 in the nuclear but not the cytoplasmic fraction using H3 Ab. However, other nuclear proteins, such as Ku, can be detected in the cytoplasmic fraction indicating the leakage of the nucleus. Now, I'm trying different lysis conditions and hope one of them will be suitable for the cell lines I'm using now (MCF7).

Yuanita

[quote name='Yuanita' date='Dec 6 2005, 08:50 PM' post='33698']
[quote name='rajgene' date='Dec 6 2005, 07:44 AM' post='33618']
Hi
You can use Ab against histone proteins in the nucleus. As far as the lysis is concerned make sure that the nuclear pellet is once rinsed with ice cold PBS once (favourably with phosphatase/protease inhibitors) so that there is no carry over proteins of the cytoplasmic fraction
Ope this helps

Thanks for the info, Rajgene.
Actually, using histone as marker might give some false results because histones are always in the pellet after separation. I always can detect histone H3 in the nuclear but not the cytoplasmic fraction using H3 Ab. However, other nuclear proteins, such as Ku, can be detected in the cytoplasmic fraction indicating the leakage of the nucleus. Now, I'm trying different lysis conditions and hope one of them will be suitable for the cell lines I'm using now (MCF7).

Yuanita
[/quote]
yuanita, i'm not sure. but i think Ku proteins are also present in the cytoplasm in normally growing cells. something to do with mitosis, or whatever.
therefore, i wouldn't be so sure of the presence of Ku in the cytoplasm has to mean that youre dealing with the leakage....