Hey /sci/, I am working on a project and would like to discuss and brainstorm a bit about certain issues and what approach is best.
This is not a "muh homework" or anything like that, just a discussion about biochemistry.
So, say that one wishes to extract an RNA-binding protein out of organ tissue, with the scope of sequencing the bound RNAs, how would one do that?
Let's say that is is for research, not clinical, purposes. So genetic manipulation is allowed.
If it were a cell culture one could create a tagged-protein via recombination and then simply use an affinity column. However is this also possible in organisms like say Drosophila or Chaenorhabditis or a similarly small model organism without using too advanced methods like CRISPR/Cas9?
Using in-vitro binding (produce the tagged protein separately, then just add it to a tissue lysate) is not a good idea since it does not allow to study the physiological conditions.
Any ideas?
>If it were a cell culture one could create a tagged-protein via recombination and then simply use an affinity column. However is this also possible in organisms like say Drosophila or Chaenorhabditis or a similarly small model organism without using too advanced methods like CRISPR/Cas9?
I did something similar to that for my senior thesis in undergrad. (Note that this was 7 or 8 years ago by now, so my memory of that project had faded a bit.) I think you're on the right path, but I doubt you'd be using an affinity column to extract protein in this system. If you're over-expressing in a eukaryotic system, say HEK cells, you'd probably do an IP. From there, you could do a footprinting assay with RNAse to identify the RNA sequence bound to your protein.
The scale that you'd be expressing protein in eukaryotic cells on would probably preclude any sort of classic purification scheme, as would be done from recombinant protein over-expressed in liters of E. Coli or insect cells.
>Using in-vitro binding (produce the tagged protein separately, then just add it to a tissue lysate) is not a good idea since it does not allow to study the physiological conditions.
I agree with this. If you take recombinantly over-expressed protein and incubate with tissue lysate, you'll run the risk of non-specific interactions with RNA or DNA or other proteins, plus the hazard of introducing any number of proteases to purified protein.