Updates on all the huntingtin samples I have made and what has been keeping me busy in the lab
Updates on all the huntingtin samples I have made and what has been keeping me busy in the lab
SGC Univ. of Toronto
Why EZHIP may be a drug target?
PFA ependymomas are pediatric brain tumors with very poor prognosis. They represent about 10% of all pediatric tumors of the central nervous system (CNS), and up to 30% of CNS tumors in children under 3. (1) 9.4% of PFA tumors aberrantly express EZHIP (aka CXorf67), a protein that inhibits Polycomb repressive complex 2 (PRC2). PRC2 Read More …
Overexpressing HTT in neuronal cells: Update
In my last blog post, I described how I am trying to overexpress HTT in neuronal cells. While the BacMAM system was easy to implement in cells such as HEK293, it proved far more challenging to express in SH-SH5Y neuronal cells. Since then, I have attempted to use the BacMAM system in two other neuronal Read More …
Testing USP5 ZnF-UBD analogues with a Displacement Assay and SPR
If you’ve been following along my open notebook journey over the last two years, you’ll be excited to hear I’ve published my work on the first antagonists of USP5 ZnF-UBD in a peer-reviewed journal. Check it out here! In a previous post, I outlined my selection of the next set of analogues in my chemical Read More …
The protein WDR41, and its role in ALS.
Amyotrophic lateral sclerosis (ALS) is a terrible disease that affects the way a person speaks, moves and even breaths, and is responsible for as many as 5 deaths for every 100,000 people over the age of 20. ALS is a progressive disease, affecting the neurons in the both the upper and lower brain way[2], as Read More …
HDAC11 cellular assay validation with catalytic mutants and inhibitors
My previous results showed that overexpression of HDAC11 decrease basal acetylation levels of exogenous histones as well as decrease the acetylation increase caused by histone acetyltransferases. In order to determine if the observed effect is caused by catalytic activity of HDAC11, I used catalytic HDAC11 mutants Y304H and H142,143A (PIMID: 30819897, 28927261) and published HDAC11 Read More …
USP5 Ubiquitin-Rhodamine110 Catalytic Activity Assay
Today’s post describes testing if ZnF-UBD ligands are able to antagonize USP5 catalytic activity using a ubiquitin-rhodamine110 (UbRho110) assay, which was developed and optimized by Leanna Smith of the Biophysics group at SGC Toronto. You can see details on Zenodo. The UbRho110 assay is a fluorescence intensity assay that monitors the enzymatic activity of deubiquitinating Read More …
Do ZnF-UBD ligands bind full length USP5?
In a previous post, I described the expression and purification of full length (FL) USP5 constructs and also determined that zinc finger ubiquitin-binding (ZnF-UBD) ligands are able to displace ubiquitin in both the ZnF-UBD and the FL protein in a fluorescence displacement assay. In today’s post, I describe using a surface plasmon resonance (SPR) assay Read More …
Project Summary: Exploration of the HTT interactome
Five months ago, I made my first post outlining the goals of my project here at the SGC. I am writing again today to update you on the progress I have made toward those goals. As a brief overview, I was working on finding Huntingtin (HTT) interacting proteins, but why would this be important? If Read More …
Expression test and purification of SETDB1 catalytic domain constructs for structural studies by X-ray crystallography.
Recombinant protein expression is a prominent molecular technique for structural biology studies. For expressing recombinant proteins, a host organism is utilized as protein production factories. To obtain the desired recombinant proteins, we first design a recombinant DNA, which involves the information of protein to be produced. Then, we clone the desired DNA fragments into a Read More …