[erv10s]

Question:

You want to study the transcriptional regulation of the "fat control" gene leptin in the brain. A putative DNA element ("upstream" of the gene) to which control proteins may bind, has the DNA sequece 5' - GATCCCATTCAGGT- 3'. Propose a method to isolate novel proteins which may bind to the sequence shown. In doing so, first consider where the proteins are likely located in the cell as well as the properties the base, sugar, and phosphate that compose DNA.



So far, I have:

- Cut pieces of brain tissue
- Lyse by grinding tissue
- Subject the mixture to differential centrifugation at low speed ( 1000 x g)
- Subject to density gradient to isolate nucleus...

Would it be better to treat the mixture with some type of detergent after lysing since the protein has to bind the DNA sequence? If so, then I would get rid of the density gradient step and go straight to organic solvent precipitation via ethanol. From this point, I am unsure how to proceed.

[Arkcon]

That's not how I would do it.  Can you try working backward -- starting with the possible protein sources, and not brain tissue?

[erv10s]

Well, the protein is involved with transcription, so it's source is the nucleus. The protein of interest should bind 5' - GATCCCATTCAGGT- 3' . So, DNA-binding protein could be purifed using affinity chromatography with the sequence being the ligand that is attached via CNBr. I still am a bit confused, but am I on the right track with this?

Thanks.

[Arkcon]

Srry ... I misunderstood.  I thought you wanted the nucleus for the DNA sequence.  OK, so, you have an affinity column prepared with the short sequence, right, and want to isolate DNA binding proteins from the nucleus?  What examples have you found regarding this sort of isolation -- which particular type of detergent etc.  Obviously, if you're too harsh, you'll denature the proteins, and they may lose binging affinity.