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It’s never too late to take another look at your data. The incredibly knowledgeable Sally Cowley suggested that I shouldn’t just think about how many cells contain any AlexFluor488 antibody after electroporation, but how bright each cell is (i.e. the amplitude of fluorescence). Looking at this instead will give me an idea of how much Read More …
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 …
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 …
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 …
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 …
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 …
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 …
…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 …
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 …
Continuing with our goal to synthesize three compounds as ALK2 inhibitors, let’s refer back to Scheme 2 in the previous post on this topic, compounds 6, 8 and 9 were synthesized in good yields (65-90%), however compound 10 could not be obtained. Different conditions were tested to induce the cyclization of the bromo-pyrazine-2-carboxamide 9 Read More …
