In which I scrape by

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.

What’s yours?

About Jennifer Rohn

Scientist, novelist, rock chick
This entry was posted in Careers, Nostalgia, Scientific method, The profession of science. Bookmark the permalink.

38 Responses to In which I scrape by

  1. cromercrox says:

    Good luck. By God, Squiffy, I wish I was going with you.

  2. rpg says:

    The thing I *miss*, mostly (and very, very oddly) is setting up 24 well crystallization plates, and loading crystals into the beam.

  3. nico says:

    For me it was everything to do with gels: loading them, casting them, imaging them… I am nostalgic about DGGE for some reason, even though I couldn’t afford pre-cast gels and had to do them all myself.
    Oh and DNA gel extraction with those spin filters. I loved that.

  4. BMLS says:

    well medical experiment , expecially with moleculer stuff like DNA.
    PCR also makes me feel like a scientist. and electrophoresis >_<

  5. stephenemoss says:

    Good luck with getting the paper resubmitted on time – though in my experience most journal editors will accept a few weeks slippage when asked politely. Have to say that nothing used to please me more than the perfect western blot. Crisply formed protein bands, not a hint of a smile on the tracks at the edge of the gel, and no smearing, bubble marks or spotty background artefacts.

  6. Poured yourself, naturally…

  7. Ian the EM guy says:

    It seems to be a theme, but gels make you feel like a proper scientist. Loading the wells, be it a DNA agarose gel or an SDS PAGE experiment, the act of loading the wells is almost the very essence of the publics perception, and therefore subconciously ours too, of what being a scientist is. It has the element of pipetting small volumes of coloured liquid from a tube and accurately placing them somewhere else, with the instant gratification of a job “well” done as you watch them sink if you do it without splurging your sample all over the place (Well done, did you see what I did with the “well pun” there?)

    This rosy feeling may in part be due to the fact that I don’t have to do it anymore, but I think I alwyas enjoyed loading gels. Casting them, no. Staining and destaining is ok but no big thrill. Blotting; Meh! Loading them however; yes.

    In the same way I’m not a huge fan of DIY, but drilling or sawing makes me feel like I’m my Dad, and not much beats that feeling!

  8. rpg says:

    Heh, Ian–I’ve had to tinker with the lawnmower a couple of times recently. There’s nothing quite like mucking around with an internal combustion engine to *really* feel like a man. I’d say like my dad, but his shtick was more working out best ways of nuking the Commies than fixing lawnmowers.

  9. ricardipus says:

    Ah, cell scraping – I myself have done it. It’s refreshingly low-tech isn’t it? In my case it was for drug binding assays and/or intracellular cAMP second-messenger assays.

    Listen to me, all pharmacology-like.

    I’ll jump on the “molecular biology and gels” bandwagon and say that my absolute favourite was good old fashioned S-35 labeled Sanger sequencing reactions – I used to be a dab hand at setting up 12 or so at once in a microtitre plate (by hand; the plate was just a useful container for 12 x 4 reactions). And the gels – glorious 14 by 17 inch polyacrylamide slab gels. If there was one thing I was really, really good at, it was pouring those things, no bubbles, just a lovely sheet of PAGE for those sequencing reactions to run through. 🙂

    Feeling a slight nostalgia for wet-lab work… ah good, it’s passed.

    Thanks for the trip down memory lane, and good luck with that re-submission.

  10. I, of course, famously sequenced more than a megabase of DNA using Sanger in grad school: Over the four years I developed a rhythm for each day: pour the gel (flawlessly) for the next morning; perform the reactions and run that day’s gel and put it on film O/N, and develop the previous day’s film, read the sequence and enter it onto the computer using the (extremely faded) G, C, A and T keys.

    I find it sobering that the entire project could have been completed in about 24 hours today.

  11. as a student lab helper, my job was to pour sanger sequencing gels and to load and run the sequencing reactions. so i guess i was pre-programmed to miss PCR and sequencing, then analysing the sequence data to make phylogenetic trees — with my data instead of someone else’s! the entire process made me feel like a scientist, and i miss it terribly. i even check out the prices on PCRs every few months, to see how expensive it might be to purchase one so i can turn the kitchen into a lab. (i already know that a sequencer and the reagents are way out of my price range, so i don’t even bother looking at those, sigh!)

  12. I too ran Sanger gels as a tech, don’t miss it
    I miss working with anhydrous HF on a high vacuum line I built myself in grad school, and synthesis in F2/HF mixtures – give me a steel reaction bomb any day, and I am a happy girl!n

  13. Ian the EM guy says:

    If I won mega millions on the lottery, I’d definitely have a lab and an electron microscope in one wing of my mansion! I haven’t decided if that is sad or not?

  14. I just loaded a gel with hot-pink beads and purple loading buffer – looked like the girl toys section of Hamley’s.

  15. You are so much more hard core than me. I am scared now.

  16. if you wish someone to pronounce this to be a “sad” ambition, you’re looking at the wrong person!

  17. Ian: whether sad or not, you’re actually surprisingly ‘science-typical’ there – see this recent post of mine.

  18. Loading polyacrylamide gels with those extra-long, bendy pipette tips. Extra points for having to do it from behind a perspex screen because the samples are radioactive (I did a LOT of EMSAs!).

    But really, there’s nothing quite like waiting for your film to come out of the processor…

  19. KristiV says:

    I can certainly empathize on the Rac assays, having tried enough of the related activated Ras assays in the not-too-distant past. Too many twiddly finicky steps. Give me hours and hours of cell culture, microdissection, and immunocytochemistry work instead. Gels and Western blots are OK, but I’d rather work with cells and embryos.

    Hope you get the data you need soon, Jenny, but I agree that the editors are likely to grant an extension if necessary.

  20. Austin says:

    You people are all a complete bunch of cell biologists…!

    Got to be taking an inverted microscope apart, cleaning its optical innards, putting it back together and seeing it working far better, for me. Also probably the only practical thing in the lab that I’m still any use for.

    Though… one of the very first things I ever did in a lab, strangely, was pouring polyacrylamide gels, though only smallish ones (EMBL Heidelberg, Spring 1980). And in another bit of my distant dark past as an embryonic biochemist (really) I used to tend to metre-high Sephadex gel fitration columns that lived in damp and smelly cold-rooms. Don’t have desperately fond memories of that, though..

  21. Heather says:

    Here – I’ll go off the bell curve, though I am still a biologist.

    What makes me feel like I am really doing work is microsurgery in embryology. A clean quail-chick chimera… grafting bits of tissue together and testing who signals and who responds.

    Someone just called the other day and asked if I wouldn’t want to put those long-neglected skills back into practice. Very tempting, but still more deadlines ahead for me, too.

    Indeed, thanks for a great read, and I hope your resubmission is considered to be even more excellent a read by the editor.

  22. rpg says:

    Pff. None of you noticed my comment about loading crystals into beams. With bare fingers.

  23. ricardipus says:

    That is impressive – but only one gel per day? 😉

    We had at one time a large light box with a pen-like stylus attached – with a spring-loaded tip. I think it was made by Hitachi, because it hooked up with the DNAsis software package. You could calibrate by tapping the stylus on the “corners” of each sequencing lane, then tap each band as you worked up the gel and it would read the bases back to you and enter them in the software. It seemed like magic at the time. 🙂

  24. ricardipus says:

    Ooo, +1 for a SEM. I’d spend all day taking close-ups of insects and things, and all night pseudo-painting them in Photoshop.

    Add me to the “sad” category please. 😀

  25. ricardipus says:

    Oi! I am a *molecular* biologist. Cell biology, what’s that then? Something to do with “proteins”, probably.

  26. ricardipus says:

    Oh, I forgot about those. Hate the things, I’d much rather use a good old micropipettor tip (the p10 or p5 variety).

    This is all theoretical of course, since I haven’t touched a pipettor in about eight years. Thank goodness.

  27. Ian the EM guy says:

    I never used the long bendy tips, and I gave up using hamilton syringes as well, as certain people in my lab kept breaking/bending/clogging them.

  28. This just in: my second attempt at the Rac assay also worked, and this time much more beautifully! I believe I am in business – so far I’m nicely on track to show a lovely trend with good error bars, looking forward to my third repeat to nail this sucker.

    I guess science isn’t always soul-destroying. 🙂

    Happy Friday all!

  29. nico says:

    If I won mega-millions on the lottery, I wouldn’t stop at the mansion. I’d buy myself a volcano back home in Auvergne and get an underground lair built, with guard cows with frikking laser beams on their head (it’s landlocked, so sharks won’t be very useful)!

  30. MGG says:

    Good luck with the third repeat.

  31. daedalus2u says:

    In reading your post, the idea of a 3-month deadline seems strange to me. Why would a journal editor impose a 3-month deadline other than because he/she can, and because it makes him/her feel like an editor by bossing around authors?

    Does the editor want good science, or do they want you to jump through hoops to meet an arbitrary deadline just for them?

    I appreciate that pondering questions like this only distract you from what you need to do.

  32. Well, there may be other, more sensible reasons, but I suspect it is often to meet some internal target for ‘time to reach print’.

    One of the things journals now compute, and sometimes quote in their in-trade advertising material, is:

    ‘(initial) submission to printed page time’; ….. or

    ‘(initial) submission to pre=print version up on journal website time’

    This is because the total elapsed time between submission and the paper being out is seen, along with the much-loathed impact factor and the speed of reviewer responses, as one of the factors that persuades scientists these days to submit to particular journals.

    Anyway, a long time in the ‘getting a revised version together’ stage can make this ‘elapsed time’ stat look rather unhelpfully long. So I would guess that a lot of journals put a bar of two, or three months, on re-submissions. If you then take longer than this they will badge the revised paper formally as a new submission – thus effectively cutting out all of the timeline BEFORE submission of the revised version from their computed stats.

    Hence, the stat for ‘time to print’ will look snappier, and the journal will look speedier and more ‘happening’, and thus more attractive to scientists with work to submit.

  33. daedalus2u says:

    Thank you Austin, now I understand, it is a way to waste unpaid reviewers’ time by treating it as a new submission in order to uselessly puff-up a misleading and gimmicky metric.

  34. daedalus2u says:

    Just another thought, if a scientist fudged their data like that, would it be considered misconduct?

  35. They often wouldn’t actually send it to new reviewers – just back to the same ones – so the waste in reviewers’ time is actually less than my comment might have suggested. But the bit about:

    ‘uselessly puff[ing]-up a misleading and gimmicky metric.’

    – is spot-on.

  36. ricardipus says:

    Depends which journal, probably. 😉

  37. Not necessarily. One sees papers all the time where arbitrary cut-offs are put on things, like:

    ‘any value of Y that was still increasing at the time we stopped measuring was taken to be 100’

    – I wouldn’t say it was a good thing to do, but provided papers are clear about what they did you wouldn’t call it misconduct.

    Similarly with criteria for excluding anomalous data – if you set them in advance, then you can quarrel with the ones authors have used, but it is not regarded a priori as deliberately deceptive.

    This is of course one of the big problems with defining misconduct in research. What is legitimate attempts to free the pattern from the underlying noise, what is ‘inept’ or ‘shoddy’ work, and what is conscious and intended deceit? It is a much harder question to answer than people outside basic research often think.

  38. Eva says:

    The two lab things that conveyed the “scientist moment” to me (and the only things I miss)
    1. Cutting DNA bands out of an agarose gel on the UV light box.
    2. Aligning the spacers in between the glass plates for the acrylamide gels.

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