Yesterday, I managed to overcome my eagerness to see huntingtin fragments on a gel and restained overnight with blue-silver coomassie to reveal fairly respectable bands, much more amenable to subsequent extraction from the gel and mass spectrometry analysis. All of the my write up for yesterday is on Zenodo.
The team at SPARC, Sick Kids (Paul Taylor and Jonathan Krieger, pictured) will now cut out the bands of interest, digest them further into smaller pieces and identify these pieces by mass spectrometry so we can work out which parts of the huntingtin protein they correspond to. Hopefully I should have back some data within the next few weeks. This should provide some good validation on the results from the previous domain mapping experiment as the huntingtin is being cut in the limited proteolysis step by 2 different enzymes with different specificities.
None-the-less, to move this project forward, I have also been designing a series of domain constructs for insect cell expression. I should have this finalised at some point today.