You are investigating YFP, a transcription factor that binds directly to DNA. You know that it recognizes the following sequences. (Only one strand of the DNA is shown, but double-stranded DNA is required for your factor to bind).
5' ATTGCATAGTATCCTATT 3'
You have purified your factor and you would like to do a gel shift assay (EMSA).
A. Describe how you would visualize your DNA for your EMSA.
You combine the protein and DNA and run it on the gel. You see the expected shift in the DNA upon addition of your factor. We will consider this as the baseline conditions.
Explain the following in both words and with figures showing your expected results:
B. What will happen if you increase the amount of your protein in the gel shift?
C. You perform the assay again and add 100 fold excess unlabeled DNA corresponding to your fragment to the baseline condition. What will happen in this case?
D. You perform the experiment again but add 100 fold excess of an unlabeled 18 base pair fragment that is not recognized by your factor to the baseline condition. What affect will this have in the experiment?
E. What experiment would you choose to do next with YFP?