Optimizing methodology for the detection of H3K27me3 levels using flow cytometry

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 …

Determining the EC50 of Tracer-6908 with ACVR1/ALK2-NanoLuc

In nanoBRET assay, a fluorescently labelled tracer binds to the ATP-binding pocket of ACVR1/ALK2. This binding brings the tracer into the proximity (<10nm) of Nano-Luciferase which is fused to the C-terminus of ACVR1/ALK2. Bioluminescence resonance energy transfer (BRET) occurs where the energy released by Nano-Luciferase is transferred to the fluorophore via intermolecular forces. Nano-Luciferase and Read More …

Visualisation of Purified HTT Proteins (2)

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 …

The power of hope and community action: FOP family and friends conference – 19 May 2018

After the disappointment of looking through ~13000 crystal drop images over the past couple of weeks and finding pretty much nothing in most of them and nothing that actually diffracted nicely in the rest, it was incredibly heartwarming and hugely inspiring to attend the FOP family and friends conference in Manchester last weekend. FOP Friends is Read More …

Screening EGFR and GAK Inhibitors in Chordoma

A major therapeutic avenue for chordoma is targeting the receptor tyrosine kinase, EGFR. Its ligand, EGF, is overexpressed as a transcriptional target of Brachyury and polysomy of EGFR’s region of chromosome 7 is frequent in chordoma. To this extent I, in collaboration with SGC-UNC’s post-doc Chris Asquith, am working on the profiling of a number Read More …

Structure deposition of ALK2 structures and structural analysis of ALK2/FKBP12 complex with PK010710 and PK012055

Deposition: In the past two weeks my work has mostly been full of dealing with structures. The first thing I needed to do was deposit some structures of ALK2 co-crystallised with three drug molecules that I had solved for our collaborators Swen Hoelder and Liam Hudson at ICR, who had been developing compounds against ALK2. Read More …

Purification and co-crystallisation of TGFBR1 (ALK5) and FKBP12 with five ATP-competitive ALK2 inhibitors – so much disappointment

As you are probably aware by now, if you’ve read a few of my blogs, we are testing ATP competitive inhibitors to inhibit ALK2 but not any of the other 6 ALK family members. Thus, most of our activity and cellular assays test ALK2 as well as ALK5 activity in order to check the specificity Read More …