Wednesday, October 27, 2010

How do I measure the liquid junction potential?

And so it emerges from the depths.....

For years innocent readers thought they had escaped...

A horror so horrible it inspires screams...of horror

It is...the...long awaited post on how to measure a junction potential!!

It's clear from the search terms that bring people here, plenty of folks are curious about this topic. The "What is a Liquid Junction Potential" post is one of the most read. So allow me to finally follow up with how to actually measure it. Although there are the standard descriptions about junction potentials in various texts and bibles of the field, a step by step 'how-to' is lacking. Seeing as I bridge these two in real life lab,why not here?

Ok, you have some internal solutions, and you want to report the correct voltages in your paper.

You've got to account for the junction potential between the internal solution and the bath solution present when you first stick in the pipette. Now, you can calculate it if you want; there's a program built into Clampex. But, are you sure that the constants are correct for what's in your solution, especially if it has something weird in it? TEA-methanesulfonate for example. Or NMDG-aspartate - a killer internal for recording sodium current by the way. Are you even sure you made the solution correctly?

It's easy, so measure it!

When I measure junction potentials, I follow the advice of my old granpappy, as discussed in E. Neher, Methods Enzymol. 1992;207:123-31.

Here's what you want to do:
1) Get your stuff together: battery powered chlorider (don't tell me you're using bleach - GAH!!), silver wire, 3 M KCl, 5+ mL of each of the internal solutions, plus a few mL of the typical bath solution, a few patch pipettes.

2) Make a 'flowing KCl' bridge, by taking a patch pipette, breaking off most of the tip, and filling it with 3 M KCl. Chloride a silver wire and use this as the bath ground. The high concentration of KCl, and the similar mobility of K+ and Cl- will help keep the junction potential at this electrode constant, even as the bath solutions changes. I fashioned a little jig to hold the bath pipette (on left below, a piece of plexiglass on a piece of white teflon).

3) Rechloride your silver wire on the headstage, fill a normal resistance patch pipette with one of the solutions. If you're measuring only a single internal/bath pair, then you can put the internal in the pipette. But since setting this up is somewhat annoying, I often measure multiple internals relative to the same bath solution. In that case, I put the bath solution in the pipette.
4) Whatever solution you put in the pipette, add it to the bath.

5) Put your amplifier in current clamp mode (slow versus fast doesn't matter), set the 'meter' to 'Vm', make sure that there's no external command signal coming into the amplifier.

6) lower pipette into solution.

7) Use the "pipette offset" potentiometer until the meter reads zero mV. Wait a minute or so; if the voltage drifts by more than a few tenths of a mV, then you might need to rechloride the wires.

8) Completely exchange the bath solution with the solution to be measured.

9) Read the meter, which shows the junction potential.

10) Replace the bath with the same solution as in the pipette, check that Vm is back close to zero. After that you can measure any other solutions.

11) Now, to get the total transmembrane voltage in your experiments, you have to add your command voltage (or recorded in the case of a current clamp expt) to the measured junction potential. NOTE: If you switched the bath and pipette locations, then you MUST reverse the polarity. So, in the 8.2 mV above, that's actually a junction potential of -8.2, and is added to the command (e.g., a step from -80 to 0 in Clampex becomes -88 to -8 mV). If you're an anal retentive scientist *whistles innocently*, then you already account for the junction potential in your voltage protocols, resulting in nice round numbers.

There you go, and hope that helps. Questions? Let em rip in the comments.

Friday, October 1, 2010

Damn I love being a scientist!

Even though most times it's difficult, experiments don't cooperate (cells, I'm looking at you), the future isn't certain, there's NOTHING ELSE IN THE WORLD I would do rather than be a scientist. I love it.

I also love the actual doing of the experiments. There are many postdocs out there who are counting the days until they can stop doing experiments (well, no one forced you to be a Western jockey), and let other people do it. Not me. I get an inordinate amount of satisfaction from craft of experiments.

So, off to patch clamping. The soundtrack to Conan The Barbarian is blasting. We'll see if the Wheel of Pain has me or the cells as its subject.

Wednesday, September 22, 2010

Sometimes being a science dad...

means leaving your experiments early because your son has been having lots of pee pee accidents, so they're worried about UTIs, and your daughter is coughing up a lung, so might have a pneumonia.


At least the cells weren't great, cause it's easier to leave them. Anyways, the kids need their daddy!

Tuesday, September 21, 2010

Falling to Autumn

Not too much blogging going on obviously. Things aren't bad, just kinda meh. Why? Let us count the ways:

1) Return of illin'. No, I don't mean harkening back to early hip hop, I'm talking viruses. Day care center strength viruses. Day cares where the vast majority of parents are health care workers, shipping their gunk into school, letting it percolate among the young'uns, whereupon it descends upon fresh victims. I've already had one GI bug (seriously, WTF is that? They never tell you that before you have kids), and we're all getting over a head cold. Luckily though, the little girl fought it off well, didn't progress to an ear infection. *knocks on wood*

*knocks on formica*

*knocks on cinderblocks*

2) Watching young scientists get chewed up and spit out by their so called mentors. I've been in this business long enough to see some ugly stuff, and I realize that's the way of the world. But why does it seem to happen to women more than men? Maybe I shouldn't let it drag on me. What's the phrase? "Not a fucking Care Bears tea party?" There, now I feel better.

3) I have to sit through 8 one hour ethics training sessions, to fulfill some new NIH requirement. This is a total waste. No one in their right mind believes this will really change behavior, do they? It's just window dressing. I have a feeling I'm gonna be a pain in the rear in this class.

4) The filament in the pipette puller just broke, so now I get to make a new program. *sigh*

Friday, August 27, 2010

Friday Morning Quick Hits

(Ed. - Sorry about the accidental posting clogging up your RSS tubes)

I am about to start patching some cells this morning. The cells have been a tad obstreperous and ornery these days. I want to warn them though, this crap's gotta stop. So here's your heads up you little buggers:

In other news, DrugMonkey is highlighting an online comment on a recent Nature paper that point to possible GlamourMagz shenanigans: did a Nature editor string along one group working to refute a recent paper, only to publish a second group's similar work (thus scooping the first group)? And was this Nature editor friends with the 2nd group? Check it out.

Lastly, your Science Soundtrack for the week:

Yes, I just compared science to S&M. Dog upon a leash indeed.

Friday, August 20, 2010

Friday Morning Science Soundtrack

To coincide with Genomic Repairman's daily music postings, and the awesome grad school blog carnival Samia is hosting over at 49 Percent, I present my own top pick on my personal Science Soundtrack. Though it doesn't actually convey my grad school experience (which was on the whole quite good), it sure nails the post-doc.

Here it is, arguably the greatest metal song ever:


A perfect meditation on the at times abusive relationship I have with Science.

Master of puppets I'm pulling your strings

Twisting your mind and smashing your dreams

Blinded by me, you can't see a thing

Just call my name, 'cause I'll hear you scream



Just call my name, 'cause I'll hear you scream



Master, master, where's the dreams that I've been after?

Master, master, you promised only lies

Laughter, laughter, all I hear or see is laughter

Laughter, laughter, laughing at my cries

Hell is worth all that, natural habitat

Just a rhyme without a reason

Neverending maze, drift on numbered days

Now your life is out of season

My fellow masochists are invited to contribute their own picks for the Science Soundtrack.

Friday, August 6, 2010

Science Dads Reporting for Duty!

ScientistMother's recent post about a new article in Science Careers (Scientist Dads Step Up by Vijaysree Venkatraman), was welcome, extending the Work-life balance theme that the bloggers at LabSpaces have been exploring recently.

Let me first make a big fat disclaimer: I don't know anything about work-life balance, because mine has been broken ever since the second baby came into our lives (hey, who knew a 4 1/2 lb preemie could bust that up?). I ain't no role model, as I'm far from doing either the work thing well, or the parent thing well. In fact, if I had to say which I was better at, right now it'd be the parent thing.

Figure 1: The Blair spawn frolick. I'm not in the picture cause Mom had to work on both weekend days. We all survived, the house survived, and I think I even got some laundry done. Can't remember if it was folded though.

Normally I don't think of blogging about my role as a parent. As most parents will tell you, parenting is by and large boring and repetitive tasks, punctuated by both incredibly heart warming and incredibly terrifying moments. So who cares when I have to stay home with a sick kid? Or that I made dinner last night - as I have just about every night for the past 11 years of our marriage? Or when night terrors in a 4 1/2 year old return us to the sleepwalking zombies of the infant stage? Or that I left the lab early to go to a parent-teacher conference at daycare? I already lived it, what's the point to discussing it further? Plus, god forbid it be seen as cookie-begging.

Well, maybe it isn't obvious that a lot of science dads are doing the hard grunt work of parenting. Maybe because there just aren't actually do do it. Or maybe those who do feel they need to hide or diminish it, for fear of not being taken seriously. In that case, having science dads talk about their involvement would help make it more normal.

And reading between the lines of the Science Careers article suggest we need to go even farther, as these quote suggest:

"...his lawyer wife, who works part time..."

"Currently, his wife stays home to care for their two young children -- "

"But women avail themselves of those [parental-leave] policies more often than men do because men fear they may not be regarded as serious, competitive scientists if they take parental leave..."

[now, each couple makes the decisions that are best for them. I get that. But the best we got from the men for staying away from work was Chad Nusbaum and his two months. Which is definitely great. Still, can't we do more? Shouldn't we?].

And some other quotes:

"Maybe pick two hours each day on Saturday and Sunday” to balance the needs of science and home life." (F*** you dude).

"Maybe you should have married a more supportive wife" (one male postdoc to another regarding long hours spent in the lab)


So, should I do more of daddy blogging? Like I said, I'm not a role model for someone doing well at parenthood and work. I wouldn't call myself a successful scientist; I'm just kinda getting by as well as I can (though things are improving). So in that case, maybe it's worse to do more daddy blogging?

Final note: we're on vacation next week, having family time on the beach. So whoever out there is reading (not that any of you lurkers would step up and respond to this - naughty naughty), no posts.

Thursday, August 5, 2010

Team Fox or Team Hedgehog?

Team Hedgehog, all the way!

In case you haven't been following, Notorious Ph.D. posted about the differences in academic approaches between “foxes” and “hedgehogs”:

"The fox knows many tricks; the hedgehog knows only one, but he does it well."

I’ll admit it, I am and have always been, a science hedgehog. There’s nothing like a nice 10+ figure paper, replete with detailed, technically exce
llent experiments, explicit consideration of other explanations (negative results, and their controls go here) to get me all hot and bothered. It’s what attracts me, what I find compelling science, and after enough time, I’ve realized that it’s not something I can easily change about myself (part of Notorious’s post concerns exactly that: changing from a hedgehog into a fox, and also how other scholars respond).

And yet, I realize the limitations of such a hedgehogia
n approach. Sometimes, a mix of approaches is required to get an answer; hedgehogs might miss this. Sometimes, you get so deep into something that you lose perspective, seeing only the trees, and not the forest. I get that. Still, there are ways as a hedgehog to evolve without completely changing species. You can be a serial hedgehog, going deep into different topics over time (I’d put my thesis advisor in this category). Or, you could be a topic hedgehog: sticking to one topic, going very deep into it, but bringing in other techniques as needed. I’d put the guy I started grad school with in that group.

But in all the discussion on this, which has largely been pro-fox, there hasn’t been much focus on what the limitations of the fox approach is. Sure, the best foxes are out there seeding fields with new approaches, new techniques, and both answering old questions while raising new ones.
There’s another species of fox too:

We all know them. Shitty experimentalists who flit from project to project, or people with a new fancy trick that they get the same answers people in the field had already gotten (BUT LOOK, IT’S PRETTIER!). Or they ignore the previous results that their "great new technique" doesn't replicate (probably because they don't really understand the earlier results, as they never engaged them seriously).

True, there’s fewer of these kind of foxes around for long, being selected against over time. In this vein, the idea/suggestion that younger scholars must start out as hedgehogs has a lot of merit. But I've seen young foxes; it often ain't pretty.

In the end, instead of pigeonholing hedgehogs as boring old, narrow minded, one trick ponies, and foxes and shallow, incompetent, jacks of all trades, I’d rather people agree that, when well done, BOTH approaches have their merit, and that it’s important to maintain a healthy equilibrium between the two.

Monday, August 2, 2010

I am the Norge Repairman of the primary literature

In one of those strange coincidences of the blogosphere, Chad (at Uncertain Principles) and Janet (at Adventures in Science - now in her new digs are Scientopia) are discussing the role of the primary literature in the sciences, at exactly the same time as I was plumbing the depths of the primary literature.

See, for the past few weeks I myself have been conducting an extensive expedition through the literature on the M current. M current stands for muscarinic current, which is a potassium current in various neurons that is closed by muscarinic agonists (among others). This reduction leads to enhanced neuronal excitability and more action potentials. The current has been most widely studied in sympathetic neurons, which do something to pumps or plumbing or something. Go ask her.

This sort of detailed review is something I’ve been meaning to do for some time, but never forced myself to start. But the life-work balance has been recalibrated recently (hence the increased posting here - more on that later?), so I’ve finally begun. I’ve been tracing back through the literature and cited references, checking reviews, and also reading what I can find about the scientists who contributed to the field. I find this a great way to engage in the literature, because tracing the development of the ideas helps .e put them in a context that I find much easier to remember. I’d hazard a guess that this would be a lot more useful to undergraduates engaged in research, than simply throwing the “primary source” of a bunch of ganglia at them while telling them to “do research.”

I find the M current story compelling for a couple reasons. The first relates to why studying the primary literature can be an important part of doing science. The excitation resulting from muscarinic stimulation was obvserved as far back as the early 1950s, yet it wasn’t until the mid 2000s that a pretty complete picture of the entire process, including receptor proteins, ion channels, signalling molecules, was developed. And in that time, as you might imagine, there were a lot of missteps, and numerous errors. That lesson, that wrong things get published all the time, is a crucial lesson. Another lesson is that science doesn’t develop as a neat and tidy, linear march to more and more understanding. Both of these are rarely discussed in science textbooks. he only counterexamples I can come up with are Newtonian versus quantum mechanics, and Lamarck’s theory of evolution). Most of the rest of science in textbooks is so boring. Message to undergrads: Science can be a whole lot more fun than that.

The second reason is that the 55 odd year trip from initial observation to final signalling molecules gives me hope on my own research topic. In my day job I’m studying how G protein coupled receptors activate a particular transient receptor potential (TRP) channel, TRPC5. But it’s been a bear, because we’ve exhausted the usual suspects, and haven’t yet nailed down the culprit. (Also, the channel is just a pain in the f***ing ass, clearly being a devotee of Marquis de Sade.) In fact, it’s entirely possible that we’re on the wrong path entirely (I said “possible”. Not “likely”). Still, seeing as this channel was only cloned in 1996, and “real” recordings of its activity date from 1999-2000, I figure we still have some time to go before making a run at the title of “Longest duration from ion channel to signalling pathway elucidation.”

I plan to do some blogging on the seminal papers of the field, as well as the its overall development.

Thursday, July 29, 2010

Good olden tyme science (now with Squid Axon videos!)

Isis posted an awesome video of physiologist John Severinghaus discussing his work on altitude, while working on White Mountain Research Station. Very interesting, and from this neurogeek's perspective, a part of physiology that I know little about.

It got me thinking about the great old experiments in my own subfield of ion channels and electrical excitability. I'm a big fan of going back and actually, you know, reading the foundational literature in the field. That's just me, I don't expect everyone to love it, but I always wonder at how clearly those greats viewed things, and how much their work shapes the later development of the field (especially with regards to what questions are considered important).

For electrophysiology, this leads to One Prep, One Prep to Rule Them All:


As most biologists likely learned at some point in school, the squid giant axon provided the system that Alan Hodgkin and Andrew Huxley used to analyze the basis of the action potential. Nowadays though, I'd gather there aren't many electrophysiologists who have actually seen the axon or it's beautiful action potential in real life. I know I haven't.

Luckily, we have our own kick ass old science videos. Back in the 1970s, J.B. Gilpin-Brown at the Marine Biological Laboratory in Plymouth, England, filmed a movie called "The Squid and its Giant Nerve Fiber". Now, the entire work has sadly been lost, but there are some parts which have been saved. These videos are available at the Bio 300 course site taught at Smith College, and they include J.Z. Young dissecting out the giant nerve (which he was the first to describe). If you're interested in this kind of stuff, I highly recommend you go check them out (Quicktime needed).

My favorite video is the one aboutvoltage clamping the squid axon. Here are some stills I captured from it:

Figure 1:The squid axon action potential. A thing of beauty, no doubt. Complete with hot oscilloscope action (yeah, that ain't digital!). Note the afterhyperpolarization.

Figure 2: Here, Alan Hodgkin, Nobel Laureate, prepares to ACTUALLY DO AN EXPERIMENT ZOMG!!! He's picking up the axon and getting ready to insert the electrode. I know, sorry, it's an old white d00de, wearing a vest and tie ferchrissakes. But I'll admit that I love his papers.

Figure 3: A family of voltage step currents, showing the early inward sodium current followed by the delayed outward potassium currents (responsible for the membrane depolarization and repolarization, respectively). IT'S EXACTLY LIKE THE DAMNED TEXTBOOKS! Hell, I think it might be the textbook figure. Note the slight deviation in the voltage clamp (pesky series resistance; now that's a post for another day), and the potassium tail currents.

Phew, ok, sorry to get all hot and bothered. Beautiful currents will do that to me. Now I need to get to my own electrophysiology. But these are awesome. Anybody got any more old science videos to share? I love this stuff.

Belatedly, and memely, yours

Earlier this month the "Who are you, what are you doing and why do you keep looking at me!!??!" meme came back with a vengeance worse than the West Nile virus fears in Eastern Massachusetts. I saw it on Drugmonkey, wherein I cringed at my old age.

Two years ago, Ed Yong at his Not Exactly Rocket Science blog (currently living here) posted this question to his readers, asking them why they come to the blog. From there it spread to various other science blogs.

Since my own return to blogging, I thought I'd ask my readers out there why they follow these ramblings, and what brings them here. What posts do you find most interesting. I'm especially interested in hearing from any lurkers out there. Besides, DM tagged people, and who am I to ignore that?

Wednesday, July 28, 2010

It starts early

The Patriarchy that is.

The other day my 4 1/2 year old son told his mother the following:

boy: "But you can't be a doctor, you're a girl, and girls aren't doctors. They're nurses. Only boys can be doctors."

Which is of course odd, because not only is his mother very much a doctor, but so are the vast majority of mothers of the kids we hang out with (as well as a large majority of the mothers at his daycare). And he had no problem saying that Daddy is a scientist, and "does science."

It's also a big odd, because when my wife was admitted to the hospital before our second kid arrived, he referred to all the nurses and doctors as "doctors" - male or female. Even though he knew what a nurse was, as his aunt is one.

So what was it that turned the tide, and sent him to the DarkPatriarchy side?

A buncha stupid flashcards at school that show a doctor as a man, and a nurse as a woman. WTF?!?

Of course his parents reacted in the way all good generally socially progressive types would: His mother gave the daycare folks a stern talking to, while his father beat him.

Case closed!

Seriously though, how much of this can we counteract in his little brain? We're just going to have to teach him to see it as well.

(Disclaimer - if you need a disclaimer to inform you which part of this post not to take literally, you really oughta move along elsewhere.)

Tuesday, July 27, 2010

juniorprof's #painresearchmatters campaign

In case any of you readers out there missed juniorprof's roaring back into the science blogosphere, I thought I'd take a moment to highlight his campaign to improve the awareness of pain and pain research, in all its facets, from the toll it takes on those who suffer from it, to current and new therapies, to new basic science. If you have a story to add to the discussion, from whatever perspective, head over to his blog to contribute. Or check out his Twitter feed if you're into that sorta thing.

From a neuroscience standpoint, I can attest to the fact that although pain is an incredibly interesting topic, it isn't up there front and center with the Big Questions of Neuroscience. Off the top of your head, how many Nobel prizes have been awarded for pain related research? I got nothing (OTOH, there's a number of ion channel and synapse related prizes I can name). During out intro to neuroscience course in grad school, I think we had one, count 'em ONE, guest lecture about pain. Try and guess how many we had about synapses, or the visual cortex? Nothing against those systems; I love them too, and some of my best friends are synaptic physiologists. It just serves to illuminate the priorities of the field.

Come to think of it, this is reflected in my own bias as well. See, during grad school I did research on nociceptors, those ornery little neurons that convey signals about injury to the central nervous system. And yet, I never did call myself a "pain researcher," nor do I know think back on that as "pain research." Why is that?

I'll admit that some of that it to prevent being labeled as "too applied" or "too disease oriented" in my research question. I liked to believe my research had broader applicability to questions of excitability and action potential electrogenesis, so I shied away from it. Now that I think about it, that personal bias is counterproductive when it extends throughout the community. I'd hazard a guess that you probably know the feeling, that applied research is somehow lesser, or done by "those clinical people (and we all know how good they are, knowwhatImeannudgenudge, aren't we great, high fives) leads to a situation like juniorprof tweeted yesterday:

"Pain #1 reason people seek medical attention but pain rsrch less than 1% of NIH budget"
(reference for that here)

That's just crazy. Crazy crazy crazy. It makes me regret how I characterized both my research and myself as a scientist.

So I'll say it loud, and say it proud, I was, and still am (for about ~35% of my time depending on which project I'm currently focusing on and don't even get me started about that last grad school paper but don't forget I had two kids so cut me some slack) a PAIN RESEARCHER!

I think in the near future I'll write up a few posts on nociceptor excitability and ion channels. Thanks for the inspiration juniorprof!

Friday, July 23, 2010

You know what else sucks?


But I'm guessing all you on Wordpress already know that.

Somehow the block quotes in the previous post are screwing up the text color. Any ideas?

Citation Classics -

Bill Hooker over at the LifeScientists feed at FriendFeed posted a link to a collection of “Citation Classic” articles, which were short reflections written by authors years after publishing a paper that were highly cited. Many of these are available for downloads as pdfs, and I find them endlessly fascinating. Sure, many of the highly cited papers are reviews, and reflections on “What I did this summer - Sat in my office and wrote a review that my secretary typed up” aren’t terribly interesting. But by and large, those about experimental papers are cool.

Sometimes these reflections really bring home how much the practice of science has changed over time. Bernhard Frankenhauser describes his paper with Alan Hodgkin on the effect of calcium on squid axon excitability (i.e., surface charge screening effects):

“The exciting [Ed. Wait, Bernie, you forgot to tell us whether that was an unintended pun or not!] and exhausting three-month period of experimental work was followed by two years of struggle with the analysis of the measurements and with the manuscript.” [EMPHASIS ADDED]

Not too much worrying about getting scooped I see. Seriously though, how the hell did this work? I always find that halfway through the analysis and initial drafts, I need to go back and do a few more experiments, either because the initial ones weren’t quite good enough (“crap, we really needed to wait 3 minutes after agonist removal to have full recovery, not the 2.5 we used”), or to follow up and extend the observations.

But they also show that somethings haven’t changed: And that means griping about Nature editors. As John Foreman puts it (writing about his paper with Mongar and Gomperts on calcium and secretion):

“Our experiments were written up and sent to Nature. The referees’ reports were both favourable and enthusiastic, but the editorial staff of Nature was not quite so keen. It took quite a lot of pushing, as I recall, to convince them to publish the paper. In the end, of course, the relented and now, with the hindsight of the Science Citation Index, I guess they are content that we fought for what we considered to be the right place to publish this manuscript.”

HAHAHAHA, take that you bastards! Are you happy with the 550 citations you got in the 14 years since we published what you thought was boring crap? How do you like your impact factor now, eh? (Foreman wrote the above in 1987; Web of Science presently reports 615 cites).

So go check em out, and lemme hear what your favorites are.
For the electrophysiology/excitability geeks out there, here’s a few worth your time::

-Hodgkin and Huxley on their 1952 paper presenting the model of action potential. I’ve cited this, have you?
-Eccles on “how my book that was cited on 9 pages of some recent other book was published right in time for me to get my Nobel”
-Toshio Narahashi on “how I brought a vial of TTX into the US and boy I’m glad there wasn’t a TSA back then”
-Denis Noble and Dick Tsien’s on cardiac pacemaker currents. (this one’s interesting because they themselves called the current a K+ current, though they were upfront in saying the reversal potential was actually off for a strongly K+ selective pore. As we now know, HCN channels are permeable to both K+ and Na+. See, sometimes it’s ok to report observations that you’re not quite sure what to do with. Somebody else will come up with an answer.

Thursday, July 22, 2010

Calcium imaging sucks...

...unless you’re interested in calcium. If you’re using it as a proxy for channel activation then it sucks.

There’s a tendency among those of us who study calcium permeable channels to use fluorescence imaging of Ca sensitive dyes as a way to assess channel activity. And sure, the degree of channel activation will determine the resulting fluorescence signal, and generally speaking more activation will lead to more signal. Plus it’s just easy to do: in a day you can try a huge range of manipulations, you’ll get hundreds of cells as a result (making those statistics sure seem impressively significant). Compare that with a good day of patch clamp where 10s of cells is a GREAT day. I get it. I’ve been tempted by that dark side as well.

Still, frankly, it sucks. There are so many uncontrolled and/or untested variables present in your typical Ca imaging run that if you’re making dose-response curves, or inhibition curves, or whatever, then really, you’re deluding yourself if you think you’re only looking at the channel. And don’t even get me started on those people who treat a given delta ratio as equivalent over the whole range of ratios. GRRR.

In that case, it’s time to suck it up and do the electrophysiology. Yeah, it’s hard, it’s slow, blah blah waa waa whatever. If you want to characterize a channel, do electrophysiology. If you want to see how that channel affects intracellular [Ca2+], do imaging. But then really, WTF are you doing overexpressing that channel in some poor cell line, sitting in the incubator, just minding it’s own business?

Wednesday, July 21, 2010

Did I really just do that?

I put a happy face emoticon in the comment line after stopping an acquisition run.


Of course, this was a cell that I left in the middle to go to CVS and pick up some stuff. It was still happy when I got back, holding current not more than 1 pA different. By that point though, I start to get the willies using any results. Too long whole-cell and weird stuff happens.

Come to think of it though, I bet if you trawled through my lab notebook, you'd find some "WTF" in there as well. Probably some much worse language to.

So, how many of you out there do the same?

Friday, February 5, 2010

Shallow thoughts

Following the latest ScienceBlogs-NatureNetwork steel cage match (start here and follow the links), in my head the Nature Network theme song is Sarah Smile, whereas ScienceBlogs's is Rock and Roll High School.