About ChIP with plant material - Does it matter if add formaldehydrate after grinding? (Jul/23/2009 )
I am interested in viral factors that cause epigenetic changes in plant genome. Recently, I was looking for some ChIP protocols.
Generally speaking, there are not big difference for most protocols to break plant cells. They use vacuum treat to infiltrate the plant with formaldehydrate solution, then grind the fixed plant in liquid nitrogen.
However, as I learn from a neighboring lab staff, her protocol has a big change in crosslinking step. She grind the sample first and then add formaldehydrate solution. It is easier for operation. And as she said grinding will not break nuclei where chromatin can be crosslinked with formaldehydrate later.
But questions still remained to me. Is her protocol feasible?
As we are not sure the nuclei are 100% intact, so the broken nuclei might release chromatin and that chromatin might bind or adhere with unrelated protein, thus creating some false results?
waiting for a warm discussion!
I wrote to an author who published a protocol. He agrees with me that it could result in false binding of chromatin with unrelated protein. But he think "however their modifications can be also OK when ChIP is directed towards proteins very tightly associated with DNA e.g. histones"
My primary concern is histone modification. So maybe the modification is a good shortcut.
However, I am still suspicious to this.