Hi Antonio,

As per my experience regarding this, i too worked with chromatin binding proteins… Most of my studies based on ChiP assay, in that my interest is to study  the Protein:DNA, so the standard protocol said mild cross link followed by sonication is depends upon the required size of DNA… 

In our Laboratory we use, formaldehyde cross link for 10min, quench with 1.25M glycine 15min, followed by sonication  6X15sec (interest of DNA size is b/w 100 to 200bps).. Its worked well for Us..

Good Luck  

Hi Panneerselvam,

Thanks for your answer. RNA isolation is the problem. Maybe an example is clearest.

Ezh2 is a protein from PRC2 complex that have, at least, three protein more, Ezh1, EED and Suz12. Many LncRNAs is described that bound to Ezh2 (that bound to crhomatin), so, if i want isolate these LncRNAs bound to Ezh2, but too at all PRC2 protein; Do i need fix cells with formaldehide to maintenance all protein complex and RNA? or is not necessary if we use non-denaturant buffer and protocol?. 

So, the problem can be the DNA bound to protein? Fix cells??

And the sonication, because can degradate de LncRNA or cut (if you want use to amplify and cloning) non fix cells??

Best,

Hi Antonio,

Got your point, here is 2 options...

1st try to use CHOPS containing lysis buffer, regular lysis buffer we use either NP-40, RIPA etc… But all detergents in that buffer may destroy the complex or change the nature of conditions (RNA, DNA and Protein). But, my experience CHOPS containing buffer much better to keep it safe our samples through out the experiment...

2nd mostly fix the cells help to either Protein; protein or Protein;DNA complexes, we recently started RNA debranching enzymes in ChiP assay, we still fix the cells and go for sonication steps..

This may help you,

Best Wishes your experiments... 

Thanks Pannererselvam. Sound good! I am trying bouth things, so i coul tell you next days. Not fix and not sonication and fix and sonication with the same buffer.

Thanks you so much,

Have a nice day!

Dear, Antonio! I'm trying to identify RNA-targets of the protein. This protein can bind DNA. So I' m curious which method did you use? Formaldehyde and sonication? And what about DNase treatment?

Thank you!