Or: Making Shit Glow Green (and Sometimes Red)
So my PhD project is focused on human disease genetics, and one of the best parts (also sometimes the worst, when it doesn’t work), is visualising proteins of interest in live cells.
Now, proteins are pretty tiny beasts, on average only a couple tens of nanometres across (yes, there are some super large ones around, I work with them too), so how would you see where they are in a cell?
Quick answer: Make them fluoresce.
Wham, bam, skeletal muscle actin (ACTA1) ma’am.
Now if you have any knowledge of molecular biology at all, more than a passing interest in science, or have at least had a casual interest in who won a Chemistry Nobel Prize recently, you should know about green fluorescent protein; or GFP for short.
Tack the coding sequence of that little helper onto the end (or before the beginning) of a gene expression construct using the SUPREME COSMIC POWER of molecular biology, and voila! You now have something that when slapped into the correct cell type will borrow some of its internal machinery and start churning out your gene of interest, plus green glowy taggy bit.
Then after an appropriate waiting period, you can bombard it with laser light and image!
Of course, this is much simplified, (super, extremely, ultra simplified) and there are caveats and pitfalls and certain circumstances where this particular method of protein localisation just won’t work, but there are ways of getting around that.
These days you can get a whole rainbow of colours, from infra-red all the way to cyan. My lab favours the EGFP-N1 and C1 variants, and dsRED2-express for the times when we want our proteins to glow red, and go faster (They don’t actually go faster).
Here’s a picture of some of my work on muscle cells, trying to express a particularly recalcitrant protein that I had a hand in identifying as a disease gene.
Seriously, it is the worst protein. It should not be doing that. Stop it, bad protein.
Stay angry my friends.