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In my continued quest to develop biophysical assays to screen compounds against USP5 Zf-UBD, I have been working on optimizing fluorescence polarization (FP) assay conditions for a peptide displacement assay. The aim was to optimize buffer conditions to decrease the amount of protein required for the assay, and to determine the affinity of a ubiquitin Read More …
Repeatability is a cornerstone of science, but cells behaviour can change quite significantly if you aren’t careful when looking after them. To ensure that I have the right number of DIPG cells to complete an important experiment, and that they’re all growing healthily, I want to record how fast they grow, and at what density Read More …
It’s important to have more than one assay in place to validate experimental results. In addition to the fluorescence polarization assay, I am also trying to develop a differential scanning fluorimetry (DSF) assay. DSF measures the interaction of small molecules to the protein as the protein progressively unfolds. The melting temperature of USP5 Zf-UBD is Read More …
Preparation of GW807982X analogues can be find in Tavares’s patent from 2004 (WO2004/35588, 2004, A1). This involves a 4‑step synthesis starting from commercially available 3,6-dichloropyridazine (see previous blog, https://openlabnotebooks.org/project-overview-work-towards-a-clk-probe). From my experience in the lab, formation of the highly nitrogenated pyrazolopyridazine 3 is not a trivial step. This is probably because the formation of the charged Read More …
Preliminary tests to check if the Fluor-de-Lys-green HDAC activity kit could be used for developing a high-throughput HDAC11 assay have been performed here using controls and recombinantly purified HDAC11. Details on Zenodo.
I haven’t done that much lab work lately as I have been working on crystal data processing among other things. However, a couple of weeks ago I purified some ACVR1/ALK2 for crystallisation for fragment screening (which happened on Friday) and for co-crystallisation with some of our compounds. The experimental details are here, on Zenodo. As Read More …
ALK2 and ALK5 are Type I receptors on the cell surface that can be activated by specific ligands (illustrated in the diagram below). Once activated by ligand, ALK2 or ALK5 interact with Type II receptor to be phosphorylated (phospho-group chemically attached to the protein). Subsequently, ALK2 phorphorylates SMAD1/5/8 while ALK5 phorphorylates SMAD2. Phosphorylated SMADs then Read More …
Towards the SGC goal of creating a kinase chemogenomic set (KCGS) we have been receiving and profiling kinase inhibitors donated by pharmaceutical companies. One such compound is PFE-PKIS 43 (Scheme 1a). We received this compound from Pfizer and it was screened against 403 kinases at DiscoverX. This compound, originally synthesized and published as part of Read More …
The early stages of a project often involve spending considerable time building tools to effectively tackle the problem. In cell biology, this often includes testing antibodies and expression constructs in your cell lines of interest. Putting in the work to properly validate these reagents early on in a study is critical. Poorly validated antibodies are, at least Read More …
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1172644 One of the main aims of my project is to understand the role of normal and mutant HTT in mammalian cells. To do so, I will be overexpressing HTT in mammalian cells using baculovirus. The use of baculovirus for protein expression Read More …
