5 replies to this topic

[OptimusBrien]

Hi guys, I have a question that hopefully somebody out there will be able to shed some light on. I have for the last few months been trying to perform some ChIP-Seq experiments and have been having issues. I am reasonably well versed when it comes to ChIP as I have been doing them for a couple of years now and have never come across a problem like this with any other cell lines. Basically, I can't burst the cells to extract the nuclei after fixation  
So I fix the cells on plates, kill the fixation, wash a few times with PBS and scrape down the cells in 1% SDS lysis buffer and then pellet the nuclei. At this point I always have nuclei. But the lysis buffer won't burst these particular cells it seems. I've tried a higher detergent concentration, i've tried douncing/syringing (alone and in combination) the samples but still it seems insufficient to burst the buggers! i have a few plans for what I am going to do next one of which is to see if I can isolate nuclei BEFORE fixation and then perform the fixation on these nuclei.....
Has anyone done this before? One fear is that something as drastic and intrusive on the cells as isolating nuclei may cause the binding of my TFs to change or may cause TFs to be removed altogether. But I'm more interested to hear if anyone has fixed nuclei for ChIP rather than whole cells.
Thanks a lot
Gerry

[Clare]

Hi Gerry,

Just out of curiousity - how do you know you haven't lysed the cells? And what cells are you using?

Clare

OptimusBrien, on 19 October 2010 - 09:14 AM, said:

Hi guys, I have a question that hopefully somebody out there will be able to shed some light on. I have for the last few months been trying to perform some ChIP-Seq experiments and have been having issues. I am reasonably well versed when it comes to ChIP as I have been doing them for a couple of years now and have never come across a problem like this with any other cell lines. Basically, I can't burst the cells to extract the nuclei after fixation  
So I fix the cells on plates, kill the fixation, wash a few times with PBS and scrape down the cells in 1% SDS lysis buffer and then pellet the nuclei. At this point I always have nuclei. But the lysis buffer won't burst these particular cells it seems. I've tried a higher detergent concentration, i've tried douncing/syringing (alone and in combination) the samples but still it seems insufficient to burst the buggers! i have a few plans for what I am going to do next one of which is to see if I can isolate nuclei BEFORE fixation and then perform the fixation on these nuclei.....
Has anyone done this before? One fear is that something as drastic and intrusive on the cells as isolating nuclei may cause the binding of my TFs to change or may cause TFs to be removed altogether. But I'm more interested to hear if anyone has fixed nuclei for ChIP rather than whole cells.
Thanks a lot
Gerry

[OptimusBrien]

Hi Clare,
So I know the cells aren't lysed just by visual inspection. If you look under the microscope nuclei look like dark/black dots while whole cells are bright/white dots. When I look under the microscope at these cells (Human mammary epithelial cells - HMECs) what I see are clumps of white cells, and a very few black dots. It seems the lysing is just really inefficient. Incidentally, I tried making nuclei and then fixing the nuclei in suspension but it didn't work!
G

Clare, on 20 October 2010 - 01:01 PM, said:

Hi Gerry,

Just out of curiousity - how do you know you haven't lysed the cells? And what cells are you using?

Clare

OptimusBrien, on 19 October 2010 - 09:14 AM, said:

Hi guys, I have a question that hopefully somebody out there will be able to shed some light on. I have for the last few months been trying to perform some ChIP-Seq experiments and have been having issues. I am reasonably well versed when it comes to ChIP as I have been doing them for a couple of years now and have never come across a problem like this with any other cell lines. Basically, I can't burst the cells to extract the nuclei after fixation  
So I fix the cells on plates, kill the fixation, wash a few times with PBS and scrape down the cells in 1% SDS lysis buffer and then pellet the nuclei. At this point I always have nuclei. But the lysis buffer won't burst these particular cells it seems. I've tried a higher detergent concentration, i've tried douncing/syringing (alone and in combination) the samples but still it seems insufficient to burst the buggers! i have a few plans for what I am going to do next one of which is to see if I can isolate nuclei BEFORE fixation and then perform the fixation on these nuclei.....
Has anyone done this before? One fear is that something as drastic and intrusive on the cells as isolating nuclei may cause the binding of my TFs to change or may cause TFs to be removed altogether. But I'm more interested to hear if anyone has fixed nuclei for ChIP rather than whole cells.
Thanks a lot
Gerry

[KPDE]

OptimusBrien, on 29 October 2010 - 06:19 AM, said:

Hi Clare,
So I know the cells aren't lysed just by visual inspection. If you look under the microscope nuclei look like dark/black dots while whole cells are bright/white dots. When I look under the microscope at these cells (Human mammary epithelial cells - HMECs) what I see are clumps of white cells, and a very few black dots. It seems the lysing is just really inefficient. Incidentally, I tried making nuclei and then fixing the nuclei in suspension but it didn't work!
G

Clare, on 20 October 2010 - 01:01 PM, said:

Hi Gerry,

Just out of curiousity - how do you know you haven't lysed the cells? And what cells are you using?

Clare

OptimusBrien, on 19 October 2010 - 09:14 AM, said:

Hi guys, I have a question that hopefully somebody out there will be able to shed some light on. I have for the last few months been trying to perform some ChIP-Seq experiments and have been having issues. I am reasonably well versed when it comes to ChIP as I have been doing them for a couple of years now and have never come across a problem like this with any other cell lines. Basically, I can't burst the cells to extract the nuclei after fixation  
So I fix the cells on plates, kill the fixation, wash a few times with PBS and scrape down the cells in 1% SDS lysis buffer and then pellet the nuclei. At this point I always have nuclei. But the lysis buffer won't burst these particular cells it seems. I've tried a higher detergent concentration, i've tried douncing/syringing (alone and in combination) the samples but still it seems insufficient to burst the buggers! i have a few plans for what I am going to do next one of which is to see if I can isolate nuclei BEFORE fixation and then perform the fixation on these nuclei.....
Has anyone done this before? One fear is that something as drastic and intrusive on the cells as isolating nuclei may cause the binding of my TFs to change or may cause TFs to be removed altogether. But I'm more interested to hear if anyone has fixed nuclei for ChIP rather than whole cells.
Thanks a lot
Gerry

After crosslinking, when the cells are lysed the plasma membrane is solubilized but the nucleus and the cytoskeleton remain intact.  Soluble cytosolic proteins may be removed but anything stably associated with the cytoskeleton will remain.  Is it possible that this is what you are seeing and not unlysed cells.

[punkyneen]

Hi,

I'm having the exact same problem.  Did you manage to find a solution?  I've done ChIP successfully before but now I'm doing it on mES cells.  I tried the usual fixation time of 8-10 mins but the cells wouldn't even swell, they looked exactly the same under the microscope.  I read in the active motif protocol that if cells are difficult to lyse it means they have been overfixed so I've reduced fixation time down to 2 mins.  Finally they are swelling but they still won't burst open.  I've also tried homogenising and syringing with no luck.  I'm not sure whether its safe to reduce fixation time any more.

Any help would be great!!!!
Thanks

[chabraha]

See KPDE's comment above.........the cells are lysed but because they are fixed the cell morphology remains generally intact. Also you can use a non-ionic detergent in the first lysis and then hit the nuclei with a 1% SDS buffer before sonication. The reason you aren't seeing the cells swell is because they are dead and their membranes no longer represent a ion/osmolyte barrier.