I have a problem with my ChIP experiments.
I am working with yeast and looking to detect a specific TAP-tagged protein on chromatin. I follow a regular ChIP protocol that used to work, but now I have trouble since I lose my protein along the way and cannot detect anything by qPCR.
I usually have 200 ml OD600=1 cultures. I fix it for 25 minutes and lyse the cells. Until this point, I have checked the presence of my protein by western blot and it is still there. But then, here come my problems. I do the sonication! First round: 15 min (30 sec ON, 60 sec OFF), then I centrifuge (15 min @ 3000 rpm), take the supernatant and do another round of sonication. I checked the presence of my protein at each step and it appears that it goes away during the centrifugation step, most of it goes in the pellet (it is a protein that is bound to nuclear envelope...). Obviously, when I do the IP, I still have some of it, but nothing compared to what I started with!...and I cannot see anything...
How could I prevent this from happening, ie still having a lot of my protein bound to DNA before I start the IP?
Thanks in advance
Protein disappears during sonication-centrifugation
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