Unfortunately when I performed N/C fractionation I assessed protein precipitation. Usually I used a hypotonic buffer to separate C from the N. On the nuclei I applied a strong saline buffer. The problem is that when I diluted this buffer to perform IP, I assessed protein precipitation which hampered my experiment.
When you dilutes the high salt buffer to perform IP, you may dilute it to 200mM rather than lower concentration by adding the hypotonic buffer slowly. I had experience that 200mM is good enough for IP of nuclear proteins. In this way you may lose less protein due to precipitation.
Another easiest way to solve this problem is to buy a kit that works for extraction of those 2 fractions.
Ok thanks...I will try it and I'll let to know soon...
But have observed if 200 mM is good enough to avoid loss of transient interaction (not covalent interaction)?
Is it normal for you that I assessed precipitation only after the use of sonication or DNAse?
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