When we IP a transcription factor in the nuclear extract, we IP the DNA-associated TF and free TF. The DNA-bound TF is supposed to be more meaningful than free TF in the nucleus and cytoplasm. (Is that right?)
I want to purify the DNA-binding form of a specific TF and its interacting proteins. Can you give me some suggestions on that? It seems very few people try to do that.
Thank you very much.
I was actually just reading something similar to this question. They used a ChIP-seq and EMSA strategy to identify the DNA bound by the TF as well as other proteins associated in the complex. You can have a look at .
This is the link to the article I mentioned: http://www.sciencedirect.com/science/article/pii/S0092867411011342
Figure 1 shows the ChIP-seq (protein-DNA) and Figure 3 explains Protein-protein- DNA (EMSA)
Good luck!
If an oligo-bound form is adequate, you can biotinylate your the oligo and pull the DNA out with streptavidin... if you want to ensure you're looking at complexes that contain your TF, then IP. Or IP, then streptavadin. You'll lose a lot, so get ready to scale up.
This maybe a catch 22 situation !! Once the cell is lysed and DNA unwound, calling things "bound" to DNA and free is somewhat a semantic thing. Many DNA complexes are also stabilized by protein-protein interactions, held together on DNA by the factor that actually contacts DNA.
Thus IP from a lysed cell is anybody's guess. HOWEVER, if you fix the whole cell thoroughly, as in the starting of a ChIP protocol, then looking at DNA "bound" complexes may be more meaningful.
I believe, partial shearing or digestion will require brief?partial DNase treatment just as in RNA-CLIP(Please refer to Jennifer and Bob Darnell's paper in Cel, 2011) followed by crosslinking.Another way I can imagine would be to test different sonication conditions.
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