A growing team of groundbreaking scientists around the world are now sharing their lab notebooks online
There are concerns that compounds that are effective in inhibiting ALK2 by occupying its ATP-binding pocket might have reduced efficacy against mutant ALK2. That will be undesirable since the compounds should also target the gain-of-function mutant ALK2 in DIPG cells. The following experiment takes advantage of the fact that Activin A activates ALK2-R206H mutant but 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 …
SNS-032 is a CDK2 inhibitor with an IC50 of 48 nM in cell-free assay and is 10 and 20-fold selective over CDK1 and CDK4 respectively (http://www.selleckchem.com/products/SNS-032.html). In my previous post (https://openlabnotebooks.org/introduction-to-my-kcgs-cdk-family-project/) I mention the aim to develop a narrow profile inhibitors for understudied kinase. To do so I have synthesized SNS-032 analogues since SNS-032 inhibits 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.
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 …
Well, it’s been another week of not too much actual lab work and quite a lot of computer work. The lab work I did do was incredibly frustrating due to spending time searching around for reagents and then having a water problem with the liquid handler that is used to set up the fine screens. 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 …
Repeatability is a cornerstone of science, but cells behaviour can change quite significantly if you aren’t careful when looking after them. To ensure that I have the right number of DIPG cells to complete an important experiment, and that they’re all growing healthily, I want to record how fast they grow, and at what density Read More …
