I’ve been pondering the theoretical maximum number of simultaneous cell biological experiments that one person can do without losing it. I’ve also been testing the theory on a practical basis – on myself.
And I can safely report that, by all accounts, I now have the answer: Twelve. Plus or minus two (around p <.01, if I ever have the time to actually calculate the stats). Very soon, two months will have elapsed since a journal gave me a three-month deadline to answer one biological question and include its favorable answer in a revised manuscript. Two months: they’ve melted away. It’s not an overstatement to say that I’ve been running myself into the ground, having come up with four different ways to answer the question and pursuing all of them at the same time to hedge my bets. I have to dash around the lab with a diary and three timers to keep track of all the many ongoing experiments and their various time-points. Just yesterday afternoon alone, I had to replate some cells and fix them at various times afterwards; set up and monitor a timelapse video acquisition; zap DNA into some cultures that had already been depleted by RNA interference; spend three hours on the confocal microscope photobleaching and acquiring images for FRET analysis; analyze and record the last seven experiments that had just come down; and lyse cells for a Western blot. Along the way I accumulated a number of slides and lysates that I didn’t had time to process further, which are currently queuing up in the fridge and freezer.
It’s got so I’m lucky if I have time to burn my tongue on half a cup of coffee somewhere in the middle of all this, and I arrive home with a brain and body that feels pummelled. Nevertheless, I’ve been enjoying the turbo-gunner mode – probably because I know an end is in sight. I’d forgotten the seductive buzz of being the first one in, or the last one out, or stopping by on the weekend, all of which bring back memories of grad school and my five years of non-stop 80-hour weeks, when the body was younger and the dreams were still untainted by disillusion.
But that’s not what I wanted to blog about. I actually wanted to encapsulate my happiness about performing a procedure that, for no good reason, I’ve fallen in love with. Of all the things I have to do to meet my goal, I am most pleased with the Rac assay: a classic, old-school biochemistry technique in which you experimentally manipulate your cells according to your hypothesis, and then lyse them and pull down activated Rac (a small GTPase that orchestrates many events upstream of actin cytoskeletal rearrangements). With this, I can ask the question, is Rac activated or not when my gene is knocked down? This is the crux of the question that the journal editor posed to me.
The assay is notoriously fussy and is spoken about in hushed whispers of dread by those in the known. It relies on a protein partner called PAK, coupled to beads, which can bind to Rac only in its ephemeral GTP-bound state, and centrifugation, which bring the beads down while the inactive Rac washes away. Ephemeral is a bit of an understatement – the fragility of Rac’s active state forms the foundation of urban lab legend. I’ve done it once so far, with success, and am looking forward to my favorite part of the assay in a few hours’ time: harvesting the cell lysates. Yes, I know this might seem banal, but I adore mashing up cells and scraping their ice-cold, gelatinous innards with a plastic windshield-wiper-like paddle into a tidy tube for processing. I can’t really tell you why, but of all the manipulations I do, this is what most makes me Feel Like A Scientist. I like it so much that I even immortalized the procedure in my first novel, Experimental Heart.
I’m sure all scientists can single out one technique that most makes them Feel Like A Scientist, that gives them that fuzzy-focus, CSI Moment.