Month: March 2016
RaptorX structure prediction of huntingtin
There is a wealth of servers, freely available online, which allow analysis of any protein sequence. RaptorX was recommended to me by Dr. Yufeng Tong as an excellent structure prediction server so I decided to give it a go with the huntingtin amino acid sequence. All of the output from this work can be seen Read More …
Huntingtin domain construct design for insect cell expression
To follow up on all of the data I have obtained from the domain mapping experiments and computational analyses, I have designed a series of expression constructs to make short fragments of the huntingtin protein in an insect cell expression system. This basically means that I will hopefully be able me to make, extract and Read More …
Huntingtin linker sequence determination by computational methods – correspondence with Alex Holehouse
I firmly believe that the more scientists discuss their data with experts both within their field and outside of it, the better! Without peer review, constructive criticism and comprehensive evaluation of the data we all generate, we can not understand its limitations nor its value to others working in the same field. In this vein, Read More …
Ontario open access: accelerating science symposium
Today I am attending and giving a brief talk at the Ontario open access symposium hosted by University of Toronto. Loads of cool and innovative open access innovators and enablers are giving talks, the keynote is by John Willinsky, Stanford University. I’ll be live tweeting the day so follow the progress through my on @labscribbles Read More …
Domain mapping of full-length huntingtin by limited proteolysis with chymotrypsin – continued
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 Read More …
Domain mapping of full-length huntingtin by limited proteolysis with chymotrypsin
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 Read More …
Mapping the domains of full-length huntingtin – next installment of mass spectrometry data
Over the past couple of days I have been continuing to work on mapping the domains of huntingtin, an important thread to this structure-function project. Whilst the modelling data I posted on Monday is super exciting to look at, protein models are very aesthetically pleasing to an X-ray crystallographer such as myself, they are just Read More …
Structure prediction analysis of huntingtin using Phyre2
The fundamental goal of this project is to try and understand more about the structure of the huntingtin protein molecule i.e. what does this long chain of amino acids look like in three dimensions in the cells of our bodies? By understanding more about what this protein looks like, we may be able to make Read More …