Mass spec analysis of SMAD1 phosphorylation by ALK2 and ALK2 GS mutants in the presence of ACVR2.

I decided to test a set of my ALK2 GS loop phosphomutants for activity. As I’ve written about previously, I needed to optimise the concentrations of receptors and decided that 60uM was the right concentration to use as showing activity with the mutants and also being workable in the assay. I set up three different Read More …

Purification of more ALK2 GS loop mutants and fresh ACVR2.

So I’m still ‘trying to catch them all’ as the young folks say (!) and building up my collection of purified GS loop mutations so that I can test them all together and start building up a picture of what phosphorylation sites are necessary for SMAD1 activation and which are not. This time around I Read More …

Purification of ALK2 and SUFU

I’ve been working recently on purifying some proteins for use in future experiments and wanted to explain a little bit about what I need each construct for as they’re all for different things. Purification, as always, is a time consuming business but vital for any biophysical experiment that I do given all my work is Read More …

Deposition of ALK2 with M4K2009

Hello folks, another day another deposition. That makes it sound horribly commonplace but actually Ros put in immense effort into crystallising and solving these proteins and it’s my job just to add the final polish (fix those last stubborn geometry errors), give it a once over to make sure there’s nothing obviously wrong, and whip Read More …

Deposition of structure of ALK2 with M4K2117

One of the big aims of the M4K project was to look at the structure of potentially interesting molecules bound to the protein ALK2 and use that to try and design better molecules that might be useful in the treatment of the rare childhood brain cancer, DIPG. Ros did a lot of work on this Read More …

More GS loop mutations – purification of ALK2 proteins.

I’ve been getting my stocks in order recently and catching up on some other work which has been both busy but not the most thrilling in terms of experimental results. As such I’ve not had much time to write up a blog post so I’ll try and get back on top of that although it’s Read More …

Purification of GS loop mutants of ALK2

…and analysing their activity! Following on from the work I talked about here I’ve been able to start looking at the influence the different phosphorylation sites have over the ability of ALK2 to activate SMAD1. Just coming through the SGC expression pipeline are several constructs containing mutations in various combinations of the sites known to Read More …

Selectivity against, and action of, TNIK kinase in relation to ALK2.

Sorry I’ve not written for a while. I had some annual leave to use up and then I’ve been helping out with a residential EMBO course that was being co-organised by one of the group leaders in my department. The course was an introduction to a wide variety of biochemical and biophysical techniques. I was Read More …

ACVR1 Crystals taken to the Diamond Light Source (Beamline I04.) – no data sets collected.

Following up on the crystals from my last post I sent them along on the last Diamond trip we had to beamline I04 but regrettably didn’t get any usable datasets. Perhaps fortunately there is a possible explanation for this that isn’t just ‘the crystals were a bit too rubbish’. We send our crystals to the Diamond Read More …

Re-Purification of ALK2 for crystallisation with different compounds from M4K.

I’ve been lucky enough to have a student (Mira) in the lab working with me for the past few weeks and so I’d like to write about the work that we’ve been doing together. We spent some time cleaning up an old sample of ALK2 that Ros had left over in the freezer to set Read More …