A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
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 …
Aside from having high potency towards ACVR1/ALK2, inhibitor compounds ideally should be highly selective and not target other members of the Transforming Growth Factor beta (TGFb) superfamily. TGFBR1/ALK5 is selected for off-target screening because of its potential role in cardiac functions. Firefly luciferase is transcribed downstream of ACVR1/ALK2 in BRE reporter plasmid and downstream of TBFBR1/ALK5 in CAGA reporter plasmid. Renilla luciferase is constitutively transcribed 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.
It’s been a little while since I posted. I’ve been really busy, but haven’t had that much to report. I’ve been doing some processing of the fragment screening data we have so far. We found one possible hit in the pocket we are looking at for allosteric ligands, which initially looked very exciting, but on Read More …
It’s usually a good idea to have more than one trick up your sleeve… So with that in mind, I’ve started developing another viability assay for my DIPG cell lines. The first assay relied on convincing a cell line that naturally grows as floating spheres to grow stuck down on the bottom of plastic wells, Read More …
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 …
