
Data
Starting new projects in huntingtin structural biology
Hi folks! This last month or so has been a bit of a whirlwind. I was ecstatic to receive the HDSA career development fellowship and we were also successful in our funding applications to the Huntington Society of Canada as well as the CHDI Foundation. There will be some slight redirection in the projects but Read More …
Huntingtin Intrabody and Nanobody Literature Review
Happy new year! I hope that you all had a very happy holidays and that 2018 is treating you well so far. To kick off the new year, I have been doing some reading on huntingtin intrabodies and nanobodies and have written this up into an informal literature review. These are small Ig domain proteins Read More …
Cryo-EM updates and more!
This past few weeks have been busy with fellowship applications, writing grants and papers so I am rather behind on keeping you updated but there has been some exciting progress: Susan Lea has made fantastic progress processing cryo-EM data of our samples and we now have a 13 A envelope of the protein: https://zenodo.org/record/841824 2. On Read More …
Huntingtin +/- DNA oligo cryo and negative stain grid set up and screening
This past week I have been working in Oxford at the Sir William Dunn School of Pathology under the tutelage of Justin Deme. We generated some grids of the samples I prepared in Toronto a few weeks ago which involve harsher grafix conditions with greater exposure of the protein sample to the cross-linking glutaraldehyde. All Read More …
Investigating huntingtin DNA binding
Studies which look for genome wide-associated risk factors (GWAS) have a shown a link between Huntington’s disease and DNA repair proteins as well as other DNA related proteins including splicing and transcription factors. Huntingtin was previously described as a DNA binding protein but I have not found studies using purified huntingtin protein and DNA samples in Read More …
Preparing huntingtin protein samples for analysis by electron microscopy
Last week, I was busy in the lab making different huntingtin protein samples for analysis by electron microscopy, experiments I will be doing with my collaborators Justin Deme and Susan Lea in Oxford this week hopefully. I have made protein samples both with and without the N-terminal FLAG tag and have also made protein samples Read More …
Purification of full-length huntingtin (HTT) constructs Q17 and Q46 from HEK293 cells
Whilst the purification of the full-length huntingtin protein from the insect cell system we have working in the lab, gives high yields so plenty of protein to do experiments with, the protein is not made in a way which 100 % represents how it is made in humans. Human cells modify the protein with chemical Read More …
Full-length huntingtin purification and cleavage of the N-terminal tag
Last week, I completed a purification of the full-length huntingtin protein Q23 from baculovirus expression system (BVES). This protein I hope to use in a series of binding assays to see if I can find any DNA or protein partners of the huntingtin protein. The current construct I have for protein expression (kindly provided by Read More …
Purification of full-length huntingtin – trials and tribulations!
I am back from vacation and time spent on other projects so now I am back in the lab working on my HD project. Unfortunately, it has not been smooth sailing, I have had numerous issues making the full-length huntingtin sample in the lab. The protein is consistently aggregating in the purification protocol but I Read More …
Making huntingtin domains and exciting EM updates
This past week I have been trying to pin down some of the problems I have been having with making huntingtin proteins. Whilst the full-length protein prep is optimised, making smaller regions of the protein has been somewhat problematic with inconsistent yields between preps. This past week with the help of our excellent eukaryotic expression system Read More …