Purification of ACVR1 for co-crystallisation with new compounds and for fragment screening crystals

I was away on leave last week, but happily, before I left, I set up 25 crystal plates of ACVR1 with various compounds. The details of the protein purification, the compounds used and some of the crystals obtained are here, on Zenodo. So I was able to at least come back to something after my Read More …

Developing an assay of viability in DIPG cell lines

Over the past few weeks I’ve been perfecting a method for screening a small set of therapeutic compounds on DIPG cell lines. Before starting I made sure to discuss the techniques used in other groups to make sure that I design an assay that is comparable to theirs, although I may make my own changes Read More …

Update on fragment screening of ACVR1 co-crystallised with LDN-193189

I’ve put the Molprobity stats for the model I have refined to run my fragment screening datasets against in Zenodo. It looks pretty good, and I was happy when the parameters finally all went into the green. I have about 90 datasets with resolution lower than 2.6 Å, so hopefully I’ll get at least one Read More …

Co-crystallisation and fine screening of ACVR1 with K62821a, K62980a, and K62981a

As mentioned in my Zenodo post here, I fine screened ACVR1 with the three compounds in the title. I got loads of hits for co-crystallisation with K62821a. I mounted almost two pucks’ worth of crystals (31 crystals) and sent them to Diamond for Monday’s run. Unfortunately, none of them diffracted better than 3.5 Å, so Read More …

Transfectability of different patient-derived DIPG and Glioblastoma cell lines

Whether or not a cell line can be transfected efficiently is an important factor to consider when designing experiments. Often times scientists would like to deliver into the cells DNA or RNA with the instructions to produce specific proteins or to delete away specific proteins. Transfection via liposome (eg: Lipofectamine 2000) is a relatively simple Read More …

Determining whether anti-phospho-SMAD1/5 and anti-phospho-SMAD2 antibodies can recognize formaldehyde-fixed epitopes

Kinase activity of ALK2 and ALK5 in the cells can be quantified using specific antibodies that bind to target proteins once they are phosphorylated. However, conventional detection in Western blot requires large amount of samples and processing time (proteins are extracted from a large number of cells, linearized, separated in electric field and blotted onto a Read More …

Efficacy of inhibitor on wild-type ALK2 and R206H mutant in C2C12 cells (by DLA)

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 …

Fragment screening data processing and fine screen design

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 …

Recording the growth of a DIPG cell line

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 …

ACVR1A purification for co-crystallisation with LDN-193189 for fragment screening

I haven’t done that much lab work lately as I have been working on crystal data processing among other things. However, a couple of weeks ago I purified some ACVR1/ALK2 for crystallisation for fragment screening (which happened on Friday) and for co-crystallisation with some of our compounds. The experimental details are here, on Zenodo. As Read More …