Month: June 2017
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 …
Cryo-EM Huntingtin Q23 (+/- DNA) sample generation
Previously I showed that, in my in vitro assay conditions, huntingtin binds a short piece of DNA. To see if this is relevant and specific, I have generated huntingtin-DNA complex samples which have been prepared for analysis by cryo-electron microscopy. I have also tried to optimized the grafix procedure which cross-links and stabilizes the protein Read More …
Preliminary interaction studies with huntingtin and PRC2
Huntingtin has been reported to interact with the PRC2 complex. From the current literature, it is unclear if this is a direct physical interaction – Pubmed IDs: 26636336, 25574027, 23756188 and 19933700. As such, this experiment aimed to examine if an interaction of recombinant protein samples could be detected using a pull-down method. Unfortunately the Read More …
Cloning, expression and purification of brand new huntingtin constructs
Nearly all experiments to date have used full-length huntingtin cloned by Ji-Joon Song and Ihn Sir Seong, who kindly shared their expression constructs with us. Since then, we have cloned our own full-length expression constructs which can express full-length huntingtin protein in both insect cells AND mammalian cells. The wild-type (Q23) N-terminally tagged vector generated Read More …