A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
We are currently working on a manuscript to characterize one of the cell lines that we established from growing patient samples. I previously discussed how we established OCI-AML-20 and I’m currently working on some experiments to complete the manuscript. One of those experiments is determining whether the CD34 expression seen in OCI-AML-20 relies on the Read More …
Hello again! I have written a summary report of how we are culturing patient leukemic cells using two patients as an example, 150333 and OCI-AML-20. The report can be found at Zenodo. We are monitoring the growth of cultured cells by looking at the expression of CD34 and CD38 markers over a number of weeks. These Read More …
In my continued quest to develop biophysical assays to screen compounds against USP5 Zf-UBD, I have been working on optimizing fluorescence polarization (FP) assay conditions for a peptide displacement assay. The aim was to optimize buffer conditions to decrease the amount of protein required for the assay, and to determine the affinity of a ubiquitin Read More …
It’s important to have more than one assay in place to validate experimental results. In addition to the fluorescence polarization assay, I am also trying to develop a differential scanning fluorimetry (DSF) assay. DSF measures the interaction of small molecules to the protein as the protein progressively unfolds. The melting temperature of USP5 Zf-UBD is Read More …
Preliminary tests to check if the Fluor-de-Lys-green HDAC activity kit could be used for developing a high-throughput HDAC11 assay have been performed here using controls and recombinantly purified HDAC11. Details on Zenodo.
The early stages of a project often involve spending considerable time building tools to effectively tackle the problem. In cell biology, this often includes testing antibodies and expression constructs in your cell lines of interest. Putting in the work to properly validate these reagents early on in a study is critical. Poorly validated antibodies are, at least Read More …
The experimental set-up and results covered in this post can be found here: 10.5281/zenodo.1172644 One of the main aims of my project is to understand the role of normal and mutant HTT in mammalian cells. To do so, I will be overexpressing HTT in mammalian cells using baculovirus. The use of baculovirus for protein expression Read More …
There have been some exciting developments in the lab since my last post…I’ve got apo crystals of USP5 Zf-UBD! Experimental details can be found on Zenodo. The apo structure, which has no ligands bound in the receptor pocket has already been solved. The purified protein sample I have been working with for USP5 Zf-UBD is Read More …
USP5 is one of many USP proteins that contains a zinc finger ubiquitin binding domain (Zf-UBD). My goal is to develop small molecules that inhibit USP5 selectively. So, it’s important to make sure that the pocket where the compound will bind is sufficiently and structurally different from other USPs. I did a multiple sequence alignment Read More …
In order to study NSD3 in the context of acute myeloid leukemia, I first need to determine the best reagents/tools. This includes characterizing model cell lines as well as antibodies that can be used for future experiments. Here I looked at the expression of NSD3 by western blotting in three AML cell lines using a Read More …
