Isolation of nuclear and cytoplasmic fractions - (Feb/15/2008 )
Hi there,
I'm currently trying to isolate cytoplasmic from nuclear fraction in the preadipocyte cell line 3T3-L1. I've found one protocol, in witch sequencial lysis is done.
The thing works fine until I have to remove the supernatant from the first separation. One of the hard thing when working with adipocyte, is that induction of differentiation turns the media very "greasy". So when I pipet out the supernatant, the pellet containing nuclei gets sucked along with the supernatant because of the "greasyness" of the solution.
I have to confess I haven't wash the cells in PBS as mentioned, but I'm not quite sure that it would make any difference.
And now the question : Does anyone have a procotol that could isolate nuclear and cytoplasmic fraction from differentiating 3T3-L1????
I'm currently trying to isolate cytoplasmic from nuclear fraction in the preadipocyte cell line 3T3-L1. I've found one protocol, in witch sequencial lysis is done.
The thing works fine until I have to remove the supernatant from the first separation. One of the hard thing when working with adipocyte, is that induction of differentiation turns the media very "greasy". So when I pipet out the supernatant, the pellet containing nuclei gets sucked along with the supernatant because of the "greasyness" of the solution.
I have to confess I haven't wash the cells in PBS as mentioned, but I'm not quite sure that it would make any difference.
And now the question : Does anyone have a procotol that could isolate nuclear and cytoplasmic fraction from differentiating 3T3-L1????
Try using a gel loading tip with a thin neck. I find this helps you avoid sucking up the nuclear pellet.
Hope that works,
Ceri
at how much g r u centrifuging after lysis of plasma membrane
i also do the cell fractionation often
after lysing cells with np 4o i cetrifuge at 1000g for 10 minutes.
I've been told to increase the concentration of Triton x100 of the lysis buffer. Maybe that'll do the trick. Thanks anyway.