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The linearity for HDAC11 activity for Boc-Lys-(TFA)-AMC substrate was monitored using the newly optimized buffer conditions. This was followed upon by Km calculation. The dataset can be viewed on zenodo.
An effect of adding reducing agent on the HDAC11 activity was monitored. Dataset can be found on Zenodo.
The pH range used earlier was expanded for further optimization to check if the activity could improve. The dataset is posted on Zenodo.
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1292884. A few posts back, I tried expressing HTT with different lengths of Q repeats in mammalian cells using baculovirus (read more here). However, expression was not very good, especially for HTT with longer Q repeats (have a look at these results here). Read More …
To understand what proteins do, scientists often test the consequence their absence has on a cell. A common method uses a cells regulatory machinery to disrupt gene expression with complementary RNA molecules, known as RNA interference. Here I used small interfering RNAs (siRNAs) to deplete NSD3 and test the effect this has on epithelial cell identity in Read More …
In my last post, I developed a low-throughput 19F NMR screening assay against USP5 Zf-UBD, where ligand binding of a ubiquitin peptide was detected by perturbations in the resonance of a fluorinated tryptophan at the binding pocket. I recently screened the first batch of compounds that Ivan Franzoni and Renato Freitas, post-docs at the University Read More …
With previously optimized conditions, the linearity range for HDAC11 was obtained and the Km for the substrate was calculated. The data is posted on Zenodo.
Hello again! In previous posts I talked about how we were using a co-culture system to grow our leukemic cells. Although this is a great way to maintain cells, it can complicate some analyses where we cannot distinguish mouse (stroma) from human (AML) cells. We wanted to determine H3K27me3 levels (the mark that EZH2 is Read More …
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1253365. Recently, I helped run some Western blots for Rachel Harding, a fellow postdoc and open notebooker at SGC (read more about her work here: http://labscribbles.com/), for a publication on a toolkit to enable people to more easily purify and study HTT (you Read More …
Full-length huntingtin protein aggregation and first attempts to co-purify HTT-HAP40 from insect cells
