I have been performing chromatin extractions followed by salt washes to identify binding of my protein of interest to chromatin. Every time, after my washes, I run the remaining chromatin extract on a gel as well to see if there was anything left after my last NaCl wash.
I have consistently seen my protein of interest there and now I am wondering if this is because I am getting actin polymerization and cytoskeleton bound protein spinning down with my chromatin. I want to treat my chromatin extract with cytochalasin D but have never done it in vitro before. I have read about it being used on live cells but not in vitro.
Can someone suggest a protocol for this? / has any experience with cytochalasin or the kind of issue I might be having?
Thanks!
What is the material you are extracting from? Cell suspension, nuclear pellet or sheared chromatin pellet? You clearly suspect that actin polymerization is happening in your starting material during your procedures. What is the rationale for this assumption? I doubt actin polymerization would be happening in the lysed environment so emphatically that it would trap your protein of interest. Of course, this assumption would need revision if your protein of interest is also a supposed interactor of actin filament.
So, would it not be better to start with a simpler assumption? That is: your protein of interest is bound to chromatin more tightly than to be washed out at 350mM. That is sort of a mild ionic strength for extraction. You could try upto 600mM NaCl--and if you are careful, you would still be able to keep the chromatin from becoming a unworkable viscous mess.
Apart from the above, your protein of interest could still be trapped by the pre-existing nucleoskeleton and membranous debris and there may never be a role for "active" actin polymerization during your procedure. I suggest you try higher NaCl washes; and, just to see if it is tightly chromatin-bound, try using MNase, DNaseI or Benzonase to test if it can be released to the supernatant.
Hi Anil , thanks for your answer. I have tried up to 500mM for this construct that I showed the picture for. It is still in the pellet. I am starting with nuclear pellet that I treat with detergent to get rid of the nuclear membrane and ER followed by sucrose gradient.
I have tried up to 1M salt in other experiments and it does become very viscous but I can still work with it as you said being careful.
Can you suggest a way to deal with the nucleoskeleton issue?
Thanks!
Hi Raj, my expertise is limited to chromatin, and am not very familiar with experiments on nucleoskeleton. If you post a more detailed account of your protein of interest, your objective and procedure, maybe it wil help finding an answer.
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