Dear all, I am going to isolate a DNA/Protein complex from insect cells. In protein purification practice, generally, we use nuclease during the cell disruption to destroy the released nucleic acid to lower the lysate viscocity problem in the subsequent chromatography step(s). Could you share your expertise of that to protect DNA within protein complex while keep the viscocity manageable for the first chromatography step? Thank you in advance.
This is an interesting question. The first though that came to mind was to precipitate the DNA/protein complex, discard the non-complexed NA and proteins which would remain in the supernatant, then resolubilize the pellet at a concentration that is easily worked with. I am not sure but I hope this article might be helpful. Have fun!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557405/pdf/emboj00307-0177.pdf
Shear with a 20g needle with 1-3 passes. Stochastics will be on your side as most DNA breaks will not include your protein.
Shearing with a fine gauge needle is a good idea. If you have some idea of the sequence that is likely to be bound, you could possible try a rare cutter restriction enzyme (eg Not 1 cut on average once every 4e8 bases) If the protein-DNA exists as a complex it may provide some protection against bound DNA cleavage. The choice of restriction enzyme will be dictated by how large a sequence is bound - if you are only looking for a promoter binding for eg, then you can use a more frequent cutter provided it does not cut in the region of interest.
Are you using "naked" DNA + protein or extracting from total genomic DNA?
I would recommend you also do a quick test run where you actually add some DNase 1 to see if there is a chance your protein will hold onto its DNA and protect it from the nuclease. I found that some of the RNA-binding proteins I was studying were holding on to their RNA like limpets. They simply wouldn't let go of it and they would protect it from any nuclease cocktail I chucked at them, which was a problem for me as I actually needed to remove it. So, adding a DNase + RNase mixture during cell lysis wasn't a problem for me. You can compare the DNA content of your protein from a DNase 1 treated prep and a non-treated prep after purification via an Abs 280/260 nm measurement.
Viscosity per se may not be a problem even if reduced by dilution. Aggregates/quaternary structure may block your desired interactions.
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