I am continuing to try and understand the domain architecture of the huntingtin protein. In a bid to validate my previous limited proteolysis/mass spectrometry experiment, I wanted to see if I could get similar proteolytically stable fragments by digesting my remaining aliquot (courtesy of the generous Stefan Kochanek and Bin Huang) of full-length huntingtin protein with a different enzyme, chymotrypsin, instead of trypsin. Using an orthogonal enzyme with a different cleavage specificity is a good way to validate the regions of protein which are truly accessible to proteolytic enzymes which therefore likely represent less structured regions of the huntingtin protein and are possibly linker regions between the more structured domains of the protein.
In an eager attempt to see what, if any, fragments are proteolytically stable, I stained the gel for too short a period of time, so it is back in the silver-coomassie stain overnight. However, some bands are visible (wahey!) around 100 kDa and 120 kDa so I am very hopeful as to what I will see tomorrow! You can read all about the details and see a picture of the gel on Zenodo. More on this in the morning.
One thought on “Domain mapping of full-length huntingtin by limited proteolysis with chymotrypsin”
First off I want to say great blog! I had a quick question which I’d like to ask
if you do not mind. I was curious to find out how you center
yourself and clear your mind prior to writing.
I’ve had trouble clearing my mind in getting my ideas out there.
I truly do take pleasure in writing but it just seems like the first 10 to
15 minutes are usually wasted just trying to figure out how to begin. Any recommendations or tips?