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I have had little success with DSF and FP assay development for USP5 Zf-UBD to identify small molecule ligands of this domain. I decided to try 19F NMR as a potential screening assay. NMR spectroscopy can be used to visualize changes in protein spectra upon addition of low complexity molecules as well as to quantify Read More …
In my last post, I determined that fluorescence polarization with different lengths of ubiquitin peptides was not a viable assay to screen compounds. I wanted to see if the ubiquitin peptides of different lengths resulted in a thermal shift of the USP5 Zf-UBD in a differential scanning fluorimetry assay. Experimental details are on Zenodo. As Read More …
Before performing the kinetic study for calculating the Km for HDAC11, it is important to know the duration of the stability of the protein under the assay conditions. This is being analyzed here.
A buffer screen at various pH was performed to pick the buffer that gives the best activity for HDAC11. The dataset is posted on Zenodo.
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1233602. Lately, I’ve been helping Rachel Harding, a fellow postdoc at SGC and one of the pioneers of the Extreme Open Science Initiative at SGC, with some HTT experiments. She is also working on HTT, and has in fact been working on Read More …
Since the activity of HDAC11 has been observed to be quite low as per the previous data, a preliminary test was performed to check any drastic increase in the activity in the presence of an additive. The data can be found on Zenodo.
The time of incubation and conc. of the developer to be used are important parameters during HDAC11 assay optimization and thus, have been considered here. The dataset is posted on Zenodo.
In previous experiments, a FITC-RLRGG ubiquitin peptide had a Kd of ~55 µM for USP5 Zf-UBD. Different lengths of ubiquitin peptides were designed to see if increasing the peptide length resulted in a gain in affinity for the protein domain in the FP assay: LRLRGG, RAHGLRLRGG, RAHGRAKHGLRLRGG. The 6-mer peptide, LRLRGG, is the native C-terminal Read More …
In my last blog post, I observed morphological changes that were indicative of an epithelial to mesenchymal transition (EMT) when NSD3short was overexpressed from a stably integrated transgene in the cancer cell line H1299. Importantly, NSD3 has been identified as amplified in a number of cancer types, including lung (Figure 1) (cBioPortal: Cerami et al., Cancer Discov. 2012 Read More …
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1218375 The etiology of HD is the expansion of Q repeats or CAG repeats in the HTT gene, which produces the huntingtin protein. This causes improper folding of the protein and leads to protein aggregation in neuronal cells, which ultimately causes the Read More …
