A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
Hi, Recently, I have started working on NanoBret PPI technology for chromatin factors and E3 ligases are here is one of the assays I developed. Background (source: PMID28179136). The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex alters chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylation function. It is composed of six different Read More …
Hi Here are the details of the assay I developed to measure interaction between USP5 and ubiquitin in cells. Ubiquitin specific peptidase 5 (USP5) belongs to the largest ubiquitin-specific protease (USP) subfamily and has a zinc finger at its N-terminal portion and two ubiquitin-associated domains in the large catalytic region that mediate polyubiquitin binding (PMID:28923280, Read More …
Hi all! Hope everyone is staying safe and practising social distancing during these difficult times. Working from home, I’ve decided to catch up on my open notebook. In a previous post, I purified various full length USP5 constructs and determined the binding affinity of ubiquitin to these constructs. I wanted to determine the stoichiometry of Read More …
In a previous post, we solved the co-crystal structure of the USP5 zinc finger ubiquitin-binding domain (ZnF-UBD) with a promising compound, UBTR012574, which had a potency of approximately 10 µM (Figure 1). W expanded the chemical series of UBTR012574 by docking analogue compounds from the Enamine REAL database and Scifinder. We decided to order 17 Read More …
Previously in our lab, Jiayan Wang et al. worked on a project related to the druggable genome. In her project, she evaluated the druggability of protein domains found in the human genome using protein structures bound to druglike ligands. The left panel in Figure 1 demonstrates her workflow to define ligand binding pockets. To build Read More …
In my previous purification of the HTT N-HEAT_81-1643 construct, I showed that an additional heparin step did not remove any traces of nucleic acid material, changed purity or oligomeric state of the sample. Additionally, my DLS results showed that this construct forms large particles in solution. Thus, the DLS results in combination with the gel filtration Read More …
A Happy New Year to all! I am back from vacation and ready to jump back into my work with USP5. Great news- we solved the co-crystal structure of USP5 with a promising compound, UBTR012574a (Figure 1) (discussed in my last post), just before the Christmas holidays. You can check out details about co-crystal growth, Read More …
In my previous post, I shared the expression and purification results of several constructs based on bacterial expression systems. Such experiments often fail as there are inherent complications with protein expression associated with bacterial expression systems. This was likely the case for most of our last batch samples. To tackle this problem, we decided to Read More …
We have used the PTMScan® Mono-Methyl Arginine Motif [mme-RG] Kit #12235 from CST in order to determine novel PRMT7 substrates. In preliminary experiments done few years ago by our pharma partner several novel potential PRMT7 substrates were discovered with the kit, including HSP70, which was further validated by different methods (https://www.biorxiv.org/content/10.1101/503136v2). In order to obtain biological replicates, Read More …
In the second purification round of N-HEAT_81-1643, I added an additional purification step with heparin resin. My aim with doing this additional step was to improve the purity of the HTT N-HEAT_81-1643 sample from nucleic acid material/other proteins. Similar experiments proved successful for full HTT+HAP40 sample (by Dr. Rachel Harding). Our results showed that the Read More …
