Biol 330- Lecture 5 (recording)- Friday September 21 , 2012.
We’re looking at transcriptional regulators, we’re looking at proteins that bind to the promoter region of DNA
and turn on its expression.
You have YFG, want to identify what upstream region is the promoter, and you want to start to identify the
protein that are transcriptional regulator of YFG.
Can we identify in this experiment upstream, we want to see if we can identify DNA that bind to the proteins
We’re looking for unique histones...
We isolated a piece of DNA, we cut it up with restrictive enzymes, and we run it on the gel
Run that DNA on gel, it migrates based on its size.
Add to the piece of DNA a nuclear extract, so whatever tissue is important for your gene (cancer, liver gene,
whatever)... isolate the cells of that tissue, then isolate nuclei, wash away all the cellular cytoplasmic proteins
we don’t need... we want the nuclear extracts, the nuclear proteins because that’s where the transcription
factors will be
Take that nuclear extract and in there, there should be proteins that bind to specific regions on that DNA and
trascription factors (their job is to attract the RNA polymerase)
Then we can see that in the gel, we can radioactively label it. We can see very small amounts of DNA
We expose it to those proteins, titrate it perfectly so it’s not overload with proteins but titrate it perfectly
We can get on average one protein binding per piece of DNA, keeping in mind it’s an