Problem in first step in ChIP - (Sep/06/2006 )
Hi,
I use 293T cells and did chip by Upstate kit. After the cells were fixed for 5, 10 and 15 min, those cells did not break in SDS lysis buffer. The unfixed cells were lysated very well. I tried 37% formaldehyde and 18.5% fresh made formaldehyde. I also tried incubating cells in lysis buffer for 2 hrs on ice or 1 hour in 37oC water bath. Some cells did break in 37oC and all cells are broken when they incubated in 37oC overnight. But this is not good.
How can I break those cells?
Thanks for help.
Sonication...
I did. I use XL2015 sonicator from Heat System. I used microtip for 0.5 ml in 1.5 ml tube. I tried 20 to 60 x 20 pulses. Sonication did break cells but the chromatin is still about 10kb.
The complete lysis of cells is not nessecary?
The complete lysis of cells is not nessecary?
Sonication...
To tell you the truth, I never had this problem. I crosslink for 10 min at 37oC and then I stop the crosslink for 5 min at 37oC with glycine. Then I wash the cells, scrape them, lyse them and sonicate. I can get a good lysis and the chromatin is around 500bp. Maybe you should sonicate longer.
I did the same as you except at 25oC. Did you check the lysis under microscope?
I did. I use XL2015 sonicator from Heat System. I used microtip for 0.5 ml in 1.5 ml tube. I tried 20 to 60 x 20 pulses. Sonication did break cells but the chromatin is still about 10kb.
The complete lysis of cells is not nessecary?
Sonication...
To tell you the truth, I never had this problem. I crosslink for 10 min at 37oC and then I stop the crosslink for 5 min at 37oC with glycine. Then I wash the cells, scrape them, lyse them and sonicate. I can get a good lysis and the chromatin is around 500bp. Maybe you should sonicate longer.
I did. I use XL2015 sonicator from Heat System. I used microtip for 0.5 ml in 1.5 ml tube. I tried 20 to 60 x 20 pulses. Sonication did break cells but the chromatin is still about 10kb.
The complete lysis of cells is not nessecary?
Sonication...
To tell you the truth, I never had this problem. I crosslink for 10 min at 37oC and then I stop the crosslink for 5 min at 37oC with glycine. Then I wash the cells, scrape them, lyse them and sonicate. I can get a good lysis and the chromatin is around 500bp. Maybe you should sonicate longer.
No... A colleague did it and it was fine for her and I relied on that...
Maybe you can check the protocol from the Farnam Lab. You can write ChIP Farnam in google and be able to find it. I adapted that one to my needs
I have that protocol.
The lysis buffer works very well for unfixed cells. So there must be something wrong in fixation step but I don't know where. 
The lysis buffer works very well for unfixed cells. So there must be something wrong in fixation step but I don't know where.
Typically for me, after crosslinking and lysis, I get large complexes consisting of nuclei and cytoskeletal elements (as well as anything else which is insoluble and crosslinked to the cytoskeleton). They're kind of like a cell ghost. Could it be that what you are seeing are these complexes and not unlysed cells.
Regarding your large fragment size, do you isolate the DNA before running on a gel or do you run the chromatin extract directly. In my experience, I get smaller fragment sizes if I purify the DNA first (presumably because it's not bound to so many proteins).
Before gel analysis, I did reverse crosslink, RNase and Proteinase K diegest, just according to Upstate protocol.
I have that protocol.
The lysis buffer works very well for unfixed cells. So there must be something wrong in fixation step but I don't know where.
Typically for me, after crosslinking and lysis, I get large complexes consisting of nuclei and cytoskeletal elements (as well as anything else which is insoluble and crosslinked to the cytoskeleton). They're kind of like a cell ghost. Could it be that what you are seeing are these complexes and not unlysed cells.
Regarding your large fragment size, do you isolate the DNA before running on a gel or do you run the chromatin extract directly. In my experience, I get smaller fragment sizes if I purify the DNA first (presumably because it's not bound to so many proteins).
what is the final concentration of the formaldehyde in your fixation mixture?