I reached a milestone, or I guess you could say a mile-PAGE today – my 700th SDS-PAGE since I started numbering them to keep track and cross-reference with my notes. My lab notes read as a history of all the SDS-PAGE (Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis) gels I’ve run so far throughout my PhD-seeking journey. And today’s mile-PAGE got me thinking about the history of SDS-PAGE itself. This technique for separating proteins by size has become a staple of modern biochemistry, but how was it that that came to be? Turns out you could find the answer in another grad student’s lab notebook – although he was beaten to reporting it by another group that figured it out a few months later… So today, in an ode to students around the world, I thought I’d tell you more about the very first SDS-PAGEs!

700 – and still going strong! Some people might see these thin slabs of gels as just another one of those monotonous things you have to do if you’re a protein biochemist. But I see each of these as a tiny molecular race in which proteins, coated with a negatively-charged detergent called SDS, push past one another (within their lane), slither through the gel’s pores and near the positively charged “finish line” at the bottom of the gel. The smaller the protein, the easier the journey, so when you stop the race on demand by turning off the electricity that was creating the charge gradient, they freeze in place. And when you stain the gel you can see where they ended up and thus, by comparing to a ladder of proteins of known size (molecular weight markers) you can tell how big the various proteins are. Additionally, by looking at the number of other bands (representing other proteins in the race) you can also get an idea of how pure the sample is.

We often take this technique for granted but it has only been around since the mid 1960s. A lot of what I’m going to tell you comes from what I learned in a really great article by Thoru Pederson called “Turning a PAGE: the overnight sensation of SDS‐polyacrylamide gel electrophoresis” published in the FASEB Journal in 2008. https://doi.org/10.1096/fj.08-0402ufm 

The way the word “protein” is used in the context of nutrition (e.g. this granola bar contains 10g of protein) might give the misperception that “protein” is a single thing. But that 10g of protein actually corresponds to lots and lots of different proteins – they have different sizes and properties and different functions. In my granola bar, those functions don’t really matter because when you eat proteins you break them down for energy and parts, but in your body proteins can do a huge number of important tasks – from helping give cells structure, to copying DNA, to making other proteins. One of the only things they have in common is that they’re made up of long chains of building blocks or “letters” called amino acids – each of which has a generic backbone which allows for linking and a unique side chain or “R group” that sticks off (kinda like a charm on a charm bracelet). 

When scientists started to kinda get an idea about what proteins are – or at least realized that they existed – the concept really was more like that “10 g of protein” in my granola bar – it was this kinda weird thing that seemed to be made up of different types of weird thing, but they couldn’t tell what was in a mix 

Early hints that different things were in these mixes were found using Teselius’ breakthrough discovery of electrophoresis, a technique whereby you use electricity to create an electric gradient where there’s a positive end and a negative end, and molecules travel towards the charge which attracts them. Positively-charged things migrate towards the negative end negatively-charged things rush towards the positively-charged end. 

If you stick “untreated” proteins in an electric field, they’ll migrate based on their natural charge – different proteins have different charges because a few of the amino acid side chains are positively-charged, a few of them are negatively charged, and different proteins have different combos of them. So you can get some separation using this “isoelectric focusing” (IEF) technique (isoelectric refers to the point at which the protein reaches the spot in the gradient where it has the same “iso” (but opposite) charge as the surroundings and thus stops moving.

This sort of charge-based separation could be done in a number of formats, including on paper (which was good for peptides (small pieces of protein)) & in columns – first sucrose (table sugar) and later gels of starch and then later the polyacrylamide we’re used to now. Except this was in columns, so think “normal gels” but in a tube. If you want to learn more about this, check out my post on the discovery of the mutation in hemoglobin which causes sickle cell disease. It’s just a one letter change, but it goes from a charged letter to a non-charged letter (E to V), so the mutated protein has a different charge than the “normal” hemoglobin, so it travels differently in IEF (and has a tendency to clump together and clog blood vessels…) http://bit.ly/paulingingram 

That was using purified proteins. But what about mixtures of proteins? This technique was important for doing some crude analyses of “this protein thing” is different from “that protein thing” but a lot of proteins have similar charges and would thus travel together instead of separating. And the technique didn’t tell you anything about “size” of the protein – yes, proteins’ size would affect their movement through a gel, but the charge would also matter and they could be working in opposite directions (e.g. a big negatively charged protein could travel faster than a small neutral one). 

What they needed was a way to give all the proteins a uniform charge so that *only* size would matter. And a way to unfold them all so that “shape” (e.g. big and floppy vs small & compact) wouldn’t matter either – just “length.” The detergent SDS does both at once – it slithers into the crevices of proteins and globs on. And since it’s negatively charged it repels itself, keeping the protein linear – and now negatively-charged in a uniform manner! But the scientists who developed SDS-PAGE as “a thing” weren’t looking to do those things (at least at first). They were looking to get membrane proteins out of membranes. And, for the same reason soaps and detergents are great at breaking up viral membranes to kill viruses and bacteria, they’re great at breaking up membranes to free the proteins in them. 

So these scientists – one group studying viruses and another studying bacteria – and both trying to analyze those microbes’ proteins, decided to give it a try. 

First (well, only really) to publish was a group at Albert Einstein College of Medicine. They’d go on to use the technique with a number of different viruses, but the first published use of the technique was with poliovirus, which has a number of capsid proteins. According to this article, it was a guy named Bill Joklik who suggested the use of SDS to a postdoc named Jacob Maizel 

The first published SDS-PAGE figure doesn’t look like an SDS-PAGE at all. Instead of seeing a picture of a square-ish gel with stained bands showing the location of the trapped proteins (bigger ones higher up, smaller ones lower down), you see a sawtooth-y graph of fraction number vs. cpm (counts per minute, referring to the bursts of radiation detected). This was how they were able to “see” the proteins using radiation – they’d given the viruses radioactive amino acids to use when making their proteins. They got the virus to shut down host protein production first, so that only the viral proteins would be radioactive, meaning they’d give off radiation (waves of energy) which could be measured by a detector. 

The type of detector they used is called a scintillation counter. I use one of these for some of my experiments. Basically you stick liquid or something stuck in liquid into a little vial and stick it in a machine and it measures the radiation in the vial. But they have a gel… So they extruded the gel from the column in fractions, ground them up and put those fractions into tubes to count (keeping track of where in the gel they came from). In science talk, “After electrophoresis the gels were fractionated mechanically by extrusion through a device (to be described in complete detail elsewhere”2) which pulverized the gel in sequential fashion. The pulverized gel was carried directly into liquid scintillation counting vials by a continuous flow of water.” They counted the radioactivity & then they graphed it. And that’s what you see in the figure. 

note: “all protein” stains were available at this time, but they were interested only in the viral proteins, so using radio labeling let them do this (if they’d labeled all the proteins they’d have gotten a much messier graph!)

They did this first with purified virus, and then in virus-infected cells and found that the virus got the cell to make other viral proteins they couldn’t see before. And by adding the labeled letters at different times in the infection cycle they were able to see that the virus got the cell to make different proteins at different times. 

They didn’t know what those proteins were of course, and they didn’t even know how big they were because they didn’t use any molecular weight standards to compare to. That would come a couple years later, in 1967 when Eladio Viñuela, working as a visiting scientist in Arnold Shapiro’s lab at NYU, published a beautiful graph showing the relationship between protein size and the distance it traveled in a gel. https://doi.org/10.1016/0006-291X(67)90391-9 

You could have found a really similar graph a year earlier in the notebook of a grad student at MIT named Grant Fairbanks. He was studying E. coli membrane proteins in Cyrus Levinthal’s lab and found that SDS did the trick (in 1965, a few months before the other guys, but no one knew). And then he found that the separation going on in the gels was based on size in 1966. But this didn’t go beyond his lab notebook and thesis dissertation. 

It’s not a completely downer of a story though because Fairbanks went on to make other major contributions (which he got his due credit for). After the e. coli stuff in grad school he went to work on proteins in the membranes of red blood cells (erthyrocytes)  – using his SDS-PAGE method of course. This was “a major development” to hematology (the study of blood) and enabled much further research. But, as he warned his colleagues who were scrimping on the SDS, you’ve gotta use high concentrations of it. At lower concentrations, he’d discovered, proteases (protein chewers), being extra hardy, would survive and feast on the vulnerable unfolded proteins!

Other major advances would come with the thin slab format – polyacrylamide gel sandwiched by glass plates (often plastic ones if you buy them pre-made). This “vertical flat sheet” format is super convenient because you can run multiple samples side by side in separate lanes. For these we have to thank a couple of New Zealand scientists, M.S.Reid & R.L. Bieleski who described “A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis” in 1968. https://doi.org/10.1016/0003-2697(68)90278-9 

a lot more on SDS-PAGE: http://bit.ly/sdspageruler 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0⠀

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