The Rules of Biochemistry

My research forte is Biochemistry. I’ve been fascinated with the molecular details of how life works for a while now. I know there are other ways of looking at the world. There are scientists out there that have different perspectives and we need all of them. But being able to break biological processes down to their fundamental components, tinker with them and see how they work is my thing. I’ve learned a lot along my quest for knowledge and have passed this on to a number of Padawan learners over the years. Today, I am passing the rules on to you dear readers. Whether you are an aspiring biochemist or not these rules will serve you well in the lab.

Here they are:

  1. Fast and cold
  2. Don’t throw anything away
  3. Don’t stop until the protocol says you can
  4. Use a buffer
  5. Molecular biology is a necessary evil
  6. Manually record your data

Rule #1: Fast and cold

The first* rule of Biochemistry is that you have to be fast and cold in the lab. Biochemistry involves ripping biological systems apart. While everything may exist happily within its larger biological context, when you start breaking cells open and mixing up contents, bad things can start to happen. While you are trying to do is purify your protein or protein complex, there are proteases waiting to chew it up once they are unleashed from other cellular compartments. You also want to keep your favorite enzyme inactive until you have purified it away from everything, concentrated it and stashed it in small aliquots for future use. If you work fast and cold, then you can prevent these technical problems of biochemical purifications. In this way, biochemists are control freaks. They only want their pet proteins to have enzymatic activity when they say so. Keeping everything on ice stops unwanted enzymatic reactions. When you can’t be as cold as you would like- then be fast.** Keep your samples in the ice bucket. Don’t leave them sitting on the bench while you check that text message. Don’t touch the tubes too much with your warm hands. I’ve been told that my “fast and cold” admonitions still haunt some of my former mentees during some protocols.

Rule #2: Don’t throw anything away

Biochemical purification procedures are often multi-step protocols. The goal of biochemical purifications is to single out your favorite protein or protein complex from all the rest of the crap within the cell. Don’t worry, there is someone else out there purifying the other components that you would otherwise throw away. Unlike what CSI or other science dramas may lead you to believe, biochemistry involves a lot of clear liquids. Even for me in the photosynthesis field where pigments abound, many solutions are clear. The rest are green. Nevertheless, my protein does not call to me, “Here am I” as I am purifying it. The fractions can all look the same. Different steps in the procedure may require you to keep and continue with different parts- top phase or bottom phase, supernatant or pellet, clear fraction or green fraction.

So, even for procedures that you have done many times (but especially for those that are new)… Do. Not. Throw. Away. Anything. Keep everything until you are sure you have what you need out of the procedure. Better yet, keep everything until the experiments are finished and the paper is written. Keep everything until the paper is published and your PhD thesis is defended. If no one is going to fuss at you too much, just keep everything until your lab needs to buy a new freezer. You get the point.

You never know if you screwed something up at some point in your procedure and you kept the wrong fraction. Keep them all and you will never stare pitifully at the sink drain yearning for the precious protein that you just accidentally threw in there. There went hours- or weeks-worth of work, lost to the building plumbing system. You will have to tell your PI or other supervisor what you have done. There may be some wrath, but mostly there will be pity and then laughter. Do the procedure again.

You never know when some of those fractions will come in handy and you need those samples. Then you will look like a genius for being so far ahead on the experiment by being able to squeeze so much data out of a single prep. Also, when reviewers’ of your manuscript suggest additional experiments, you will be ready for them. Of course, this all depends on a sub-rule of rule #2 “label everything.” All of those extra samples don’t mean much when you can’t remember what was in each tube (remember they are all equivalently clear or maybe green). So write something more specific than “1” or “A”. Use the fine point sharpie and include a date. Ideally, you would also have a coordinately meticulous lab notebook documenting all samples, contents and relevant information.

Rule #3: Don’t stop until the protocol says you can

Rule #3 is just another way of telling you to obey rule #1. I know this multi-step procedure will be a pain in the rear, but you can’t just stop at any time and expect everything to be OK. The protocol should say when you can stop or pause without ruining the whole thing. No, you probably won’t get to eat lunch that day. I know there’s a free pizza lunch at the seminar, but you will have to ask another student to grab slices for you. This brings up another good point- manage your time so that your procedures do not interfere with free food opportunities. Don’t ask Dr. PhD or others in the lab if you can stop at other points. There are only two answers to that question and you won’t like either one: No, you cannot stop or the prep will be ruined OR No one has ever tried it and your prep will likely be ruined. Occasionally, laboratory laziness will result in a better way of doing your biochemical prep by introducing a newly discovered convenient stopping point (make sure to scribble this in the lab protocol and your notebook), but this is generally rare. Just do the protocol as it has been developed and do it that way every time.

Rule #4: Use a buffer

I feel like I have to explicitly say this since it came up in a lab conversation not that long ago. For every biological experiment there is to do in the lab, you will be using a buffer solution with the appropriate amount of salt and buffering agents to keep things within a specific pH range. Water may be the universal solvent and otherwise critical for life, but biology has additional constraints and demands. In order for biological samples to function, they need certain ions and they need to be kept within a pH range. No, you cannot just use water. Don’t make me roll up the newspaper to scold you again. Go sit in the corner and think about what you did. When you have identified the appropriate buffer to use, you may re-join your lab mates back at the bench.

DC comics 1965

DC comics 1965

Rule #5 Molecular biology is a necessary evil

There may be some experiments that you can do working with purely ‘wild type’ or unaltered organisms. In my field, you can grind up FreshExpress spinach from Walmart to have all the PSII you could want for some experiments. However, many of the questions scientists are trying to answer in today’s laboratories require a certain amount of molecular biology work to engineer the necessary biological samples. Even though it is tedious drudgery, grab those micropipettors with gusto and resolve to get the molecular biology work done. In larger labs, you may be able to con other students or technicians to do this for you, but I wouldn’t always count on it. To be a successful biochemist, you will have to be an adequate molecular biologist. So get someone to teach you all of the voodoo related to those techniques.***

Rule #6 Manually record your data

All scientists know that it’s mandatory to keep a laboratory notebook, but many only put in bare minimum effort in properly recording experiments and data. Many scientists have learned the hard way to be meticulous. Ideally, your notebook should contain a detailed protocol, notes on what actually happened and the data collected. You won’t remember what you did. Be detailed. The person coming behind you months or years later to repeat the experiment just might be yourself. Save your future self weeks- or months-worth work by taking a few extra minutes to write down details in full sentences. At least tape in that precious paper towel you wrote down that gel protocol on. All those x-ray films of precious blots that were consuming your life 6 months ago, you won’t recognize them and they’ll all look the same if they aren’t labeled. Yes, you have to write it down somewhere. I’m a fan of keeping a manual hard-copy notebook, but I know times are changing and electronic data management systems are available. I’m still sufficiently paranoid to warn you to keep hard copies of everything, but you have to at least keep multiple back-ups of data that must be stored electronically. Inevitably, you’ll find yourself in lab meeting with your advisor asking you pressing questions as to which sample is in which lane on a gel you did two years ago. If you can’t answer definitively, you’ll be spending your weekend repeating that experiment instead of other things you should be doing like new experiments or applying for jobs.

If you follow these rules, then you too can be a successful biochemist. They are universally applicable, no matter your particular research interest. There may be other lesser commandments, but these are written in stone. Feel free to comment below with more, if you think of any I haven’t mentioned.

Johnna

* I would also argue this is rule #2 as well, but you get the point.

** In the case of photosynthesis research, you may also want to be dark as well. Generally, biochemists strive to keep their enzymes inactive during purification so they won’t be damaged or modified during the procedure. Cold temperatures and the absence of the reactants for the enzymatic reaction make this possible. The reactants for my favorite enzyme (PSII) are water molecules and light. So for PSII purifications, you have to be fast, cold and dark.

*** Molecular biology has its own set of rules, that maybe one day I will write a post on. It involves much more sorcery and religious faith than biochemistry.

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