we know there is conserved DNA binding domain, but no information was available about exactly what DNA sequence it might be recognizing.
How would we identify the DNA sequence this protein might be recognizing?
Also how would we confirm that the protein is indeed binding to this sequence? And What type of controls would we use to further verify the results?
For well defined DNA binding patterns, you should have the sequence that can be bound to the protein in question.
Once the sequence deduced, you could:
Start by a molecular docking approach to see if the DNA fragment with the deduced sequence can effectively bind to the protein (dry lab approach with bioinformatics);
Then, you would have to synthethise a DNA fragment with the in silico tested sequence (easy or not?, it will depend on the sequence length) and test if it binds to your purified protein, which basically should consist of mixing both, then applying the right thermodynamic conditions that would allow the interaction (it will depend on the DNA sequence, and wether it is ssDNA or dsDNA), then wash the excess that didn't bind, then again ellute the DNA (in order to recover what theoreticaly was bound to the protein, and amplify the elluted DNA/ use a classic Agarose gel electrophoresis (conditions should be accroding to the DNA fragment size) to see if it is there (which would mean that the elluted fraction contained indeed the DNA fragment) (wet lab approach).
Have a look at this conversation as well, it might be helpful, as they gave precise techniques for such question.
Good luck!
Try Ensembl , add your DNA sequence and you will get a map of DBD along with protein expression.
Hi Hyungji Lee, you can try this new high through put ProNA2020 tool developed by JiajunQiu et al 2020 which just got published.
https://www.sciencedirect.com/science/article/abs/pii/S0022283620302035
You can use the online version as well via:
https://www.predictprotein.org/
- Badges
- Science method