Sunday, 24 February 2013

This week in the lab (part 2)

So I left you all on a bit of a downer last time. Trust me, it was worse for me and I had to wait 3 days for some positive news.

On Monday I sent the gel with SILAC samples on for mass spec. analysis. SILAC sounds terrifying but it's really very straight forward in principal. The key thing is that arginine and lysine can be produced as heavy isotopes - the amino acid is the same it's just that they are made from heavy stable isotopes eg deuterium, 13C and 14N. If you grow your test cells in heavy media and control cells in light media then you have a way of telling them apart (with mass spectrometry). Here's a diagram that I hope illustrates the experiment.


I was really lucky in that I sent my sample when there was no backlog of samples and got my results back on Friday morning, rather than in 8 weeks time.
I (or more accurately in conjunction with colleagues more familiar with this interpreteting) need to pick through the results tomorrow. An exciting hit is for a protein connected with a human neurodegenetative disease (ND)
Now, it's well known that you're only ever one experiment away from destroying your project but this is exciting as it actually gives me something to work with and that's exactly what I needed.

I think it's called "hope"?

The fact there may be a potential link with a human disease is a generous dollop of icing on the potential cake.

The other potentially good news is that the only mutant I have, the viable one, is the mutant for this gene. It could well be that I haven't even spotted any movement problems in the mutants and I certainly haven't been looking at their brains. Now I have a reason to look closer. It might also have no phenotype in isolation.
The first priority is to confirm the interaction which I can do in several ways, such as;

1) Confirm I can pull ND protein down with "MyProtein". This only confirms a physical interaction though.

2) Confirm a genetic interaction. There are models of the ND in flies and I can use this model to see if "mygene" mutant modifies it. This would be even better as it would confirm there's a physiological interaction and whether it is a positive or negative regulator of the ND.

If test number two works then things are looking up. If not, I may be back to square one or looking at some of the other hits from the interactome. Let's see what the next experiments bring...

EDIT: Censored on the off-chance it's giving too much away. Damn paranoia about being scooped, but you never know. It costs people their jobs so I shall remain secretive until it turns out to be a waste of time, I publish it, or someone scoops me anyhow.

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