A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
In my last post, I analyzed a structure of USP5 zinc-finger ubiquitin binding domain with a cysteine-glutathione disulphide bond. These crystals were not suitable for ligand soaking due to the formation of a disulphide bond at Cys195 with a glutathione additive that occupies the ubiquitin binding pocket in the adjacent molecule in the crystal lattice. Read More …
Previously, we established that OCI-AML-20 requires stroma to maintain CD34 expression (https://zenodo.org/record/1187083#.Wr5-1C7wbIV). In this experiment, we wanted to see whether those cells grown in suspension are able to regain CD34 expression when cells are reintroduced to stroma. We find that OCI-AML-20 regains CD34 expression when put back on to stroma (either MS5 or OP9). Full Read More …
In a previous post, I determined a crystallization condition that produced nice 3D crystals of USP5 Zf-UBD that diffracted well. Rachel Harding, a postdoc at the SGC collected data images for these crystals and solved the structure using computational techniques. You can see the solved crystal structure and a more detailed analysis of the structure Read More …
I wanted to determine whether USP5 Zf-UBD is a monomer or a dimer in solution as some known USP5 structures have shown that there is a cysteine surface residue that is not involved in Zn-coordination but forms disulfide bonds (2G43, 2G45, 3IHP). I used size exclusion chromatography with multiple angle light scattering (SEC-MALS) to determine Read More …
A follow-up to check the increased fluorescence signals at 0 min of the HDAC11 reaction with its substrate was performed. The data is uploaded on Zenodo.
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1202292 In my previous post, I used baculovirus to overexpress HTT in mammalian cells. Although I managed to overexpress HTT, this HTT is actually FLAG-tagged, and very strangely, I could not detect FLAG! Let me first explain what a FLAG-tag is. FLAG is Read More …
Recently, a study of cancer-focused protein-protein interactions identified a direct interaction between NSD3 and the oncoprotein Myc in the non-small cell lung cancer cell line H1299 (Li, Z et al. 2017). Importantly, the study found that increased NSD3 expression levels can activate Myc in H1299 cells as measured by an E-box luciferase reporter. However, the functional significance of Read More …
Using Boc-Lys-(TFA)-AMC substrate, a preliminary time-course study was initiated to determine the appropriate HDAC11 concentration and the time to be used for the reaction (which are essential parameters to perform Km calculations later). Experimental details are on Zenodo.
In a previous post, I outlined initial experiments to develop a differential scanning fluorimetry (DSF) assay for USP5 Zf-UBD where I measure the resistance of USP5 to thermal denaturation as small molecules bind and stabilize the protein. I previously could not observe any significant change in the melting temperature of USP5, even using a ubiquitin Read More …
In order to develop an assay for HDAC11, a kit-free method is being attempted wherein Boc-Lys-(TFA)-AMC, a previously reported substrate was used to check the activity of HDAC11 in a preliminary experiment. Dataset on Zenodo.
