Well, my last post on biological systems and their ingredients really touched a nerve (see here for an example). I guess I should, um, clarify my position before the leaky bottles of beta-mecaptoethanol start arriving by FedEx.
I already knew the reasons for several of the components I spoke about - EDTA, for example. And I realize that there's a reason for everything that's in there. But what throws me as a chemist is that some of these recipes seem to be handed on "just because they work" Does a particular enzyme prep need EDTA in it or not? Many times, no one checks, because it probably won't harm things and it's better to be on the safe side, so in it goes. It may be hard for a biologist to understand how odd that feels to a synthetic organic chemist, but I can tell you for sure that it does.
One of the commentors to the last post brought up an important point: biologists optimize for the function of a system. And that often means having a lot of buffers, chelators, cofactors, adjuvants, reducing agents, and chaperones floating around in there with your protein of interest, to keep it thinking that it's still in some kind of cellular environment, thus putting it in the mood to do what it's supposed to be doing. There's no point in trying to see how minimal you can make the system if it's working the way you want it to already.
But we chemists are minimalists. We optimize for the function of a system, too, but in our case, purity is usually a good first variable to tune up. The cleaner everything is in our reactions, the better it generally works. That means pure, distilled solvents, with no water in them. It means an inert gas atmosphere, so there's no reactive oxygen around. And it means that your starting materials and reagents should be as clean as you can practically get them, because when there's two percent of this or five percent of that in the flask, things often start to go wrong in unpredictable ways. When a reaction wipes out on us, the first thing we check is whether everything was clean enough.
So you can imagine how biology looks to an organic chemist, whose ideal reaction is a clear solution in a clear glass flask, with one pure solvent and two pure reactants cleanly converting to only one product. Biological systems, to us, look like trying to do science by adding squirts of barbecue sauce to bowls of beef stew. Of course, as the biologists know, the stuff in those bowls was derived from stew (and worse), and was born to the stuff. It won't work unless things achieve a certain level of stewiness, and the surest way to kill it would be to turn an organic chemist loose on it to clean it all up.
1. Hank on June 19, 2006 9:13 AM writes...
And thinking back to my days at the Molecular Biology bench, one of the more common ways to optimize/clean-up/shorten a protocol is to forget one of the steps or reagents in a recipe and carry on to see if it still works. Common example: most plasmid alkaline-lysis minipreps begin by having you spin down the bacteria and then bring them up to volume with DI water and a dash of lysozyme. What happens if you space out the lysozyme? It works just fine. Lysozyme is completely unecessary in the quest to isolate plasmids from bacteria. But because it works if it's in there it stays in the recipe.
Permalink to Comment2. JSinger on June 19, 2006 9:26 AM writes...
Lysozyme is completely unecessary in the quest to isolate plasmids from bacteria.
I haven't done minipreps in ages, but seem to recall that I'd learned to skip the lysozyme early in grad school. In general, Maniatis and The Huge Red Binder Of Cloning contain a lot of steps that are either no longer necessary with modern reagents or were never necessary in the first place.
That said, IIRC you'd sometimes get restriction enzymes that didn't like plasmid DNA that hadn't been adequately lysozymed...
Permalink to Comment3. SRC on June 19, 2006 12:00 PM writes...
In grad school I took a course in enzyme purification where we each isolated and purified an enzyme from a bacterium from which it hadn't previously been isolated, so the literature contained suggestions but not a recipe for that particular enzyme.
Consistent with your observation about chemists and minimalism, Derek, the first thing I did was to systematically leave out a component at a time to find out what was necessary and what wasn't. (EDTA was an early casualty.) I think the others thought that that was silly, whereas I had the impression that they would have been happy to blow their noses into the preps if they thought that might help.
Permalink to Comment4. NJBiologist on June 19, 2006 5:14 PM writes...
There's an aspect of organizational inertia to be considered here... people take little notice of a failed experiment that was done according to the last guy's protocol, but even if you've had several run great with changes, the first one that doesn't will bring a chorus of "I told you so"s.
Permalink to Comment5. NJBiologist on June 19, 2006 5:16 PM writes...
There's an aspect of organizational inertia to be considered here... people take little notice of a failed experiment that was done according to the last guy's protocol, but even if you've had several run great with changes, the first one that doesn't will bring a chorus of "I told you so"s.
Permalink to Comment6. Courtney Hodges on June 19, 2006 7:37 PM writes...
Hi Derek, thanks for linking to my post! I enjoy your blog quite a bit, so hopefully you aren't too offended. I work in a biophysics lab with a few physicists who tend to have a similar lack of comfort in the stews and brews we use to make our proteins.
You are absolutely right that we do things certain ways because we know they'll work that way... but a good biochemist will know what every single reagent is doing in his/her buffer, and how much of it is needed for the purpose at hand!
Permalink to Comment