Expect a big downpour because the bumbling biochemist’s forecasting some SDS-PAGE gel casting! SDS-PAGE is a technique we use (A LOT!) to separate proteins by size using electricity to send them swimming through a Gel mesh made of PolyAcrylamide & today I was teaching our new lab tech how to put the Poly in polyacrylamide. So I thought – why not teach some more people. A lot of labs buy these premade – which makes sense if you don’t use them often, but we use a lot! (I started numbering my gels for cross-referencing with my notes and I’m at 445…) Even if you never use them or get them premade, I hope you can appreciated the *radical* awesomeness of how they’re made.
Feeling all alone? Have a lone electron you can loan? In a radical polymerization ride, the oxidant APS helps put the poly in polyacrylamide! When we run an SDS-PAGE gel to separate proteins by size, we use a reducing agent to break up (reversible) cross-links within the protein. But when we actually make the gel to use, we use an oxidizing agent initiate a chain reaction that forms (stronger) cross-links in the gel. And, it’s pretty *radical*! I hope you’re *free* for a chat!
SDS-PAGE stands for Sodium Dodecyl Sulfate – PolyAcrylamide Gel Electrophoresis and it’s a way to separate proteins by the lengths of their amino acid chains. The SDS part refers to the detergent you use to unfold (denature) the proteins so their “shape” doesn’t affect how they run, just their length (no unfair advantages!) and coat them with a negative charge before you use that charge in Electrophoresis to send them swimming towards a positive charge through a Gel made up of Acrylamide that’s linked together (Polymerized) into a 3D mesh. Longer proteins get tangled up in the mesh more when they travel, so they get slowed down and when you turn off the power source, removing the positive charge, the proteins lose their incentive to move and get stuck. Then you dye them with a protein-specific dye to see where they are. More here: http://bit.ly/2GZc3tG
A POLYMER is is just a long chains of similar repeating subunits. And those subunits can be all sorts of things – they just all need to have a linkable backbone. Individual subunits are called monomers. The process of linking them is called polymerization, and the end product is called a polymer.
Your body’s filled with polymers – DNA & RNA are polymers of nucleotides & proteins are polymers of amino acids. Those are more complicated to put together (at least correctly) so your body has complex mechanisms for doing so. But polymers are found all over the place outside of your body as well, and some of these can form a gel! A GEL is an “infinitely” interconnected (like 7° of Kevin bacon) polymer mesh containing water
⚠️ POLYMERS are *different* from GELS 👉 Polymers can be *components* of gels, but NOT all polymers form gels 👉 it’s only when individual dissolved strands come together & interact to form a mesh that you get a gel 👍 When we make agarose gels to look at DNA fragments, we are NOT doing any polymerizing, just gel-ifying. We start from the polymers. More here: http://bit.ly/2Rztc4L
But when we make polyacrylamide gels we ARE doing polymerizing – we start from the monomers. Lots of labs buy pre-made gels, but we go through SOOOOO many of them that it’s much cheaper and more practical to make our own.
Thankfully, unlike when we make the polymers of nucleic acids and proteins in our cells, where accuracy’s key, when we are making gels for electrophoresis we don’t need things to be “perfect” – we just need a mesh that holds water (is a gel); has pore sizes that proteins can travel through (though not too easily); and has relatively even pore sizes throughout the gel (is homogenous) so that the protein’s assigned “lane” doesn’t matter.
We do the polymerizing by starting a radical chain reaction. The electrons of atoms generally like to hang out in pairs – these pairs can be shared by 2 atoms (in which case we call it a covalent bond) or held by 1 atom (in which case we call it a lone pair)
Sometimes, instead of a lone pair, an atom has a lone “loner” – a single electron hanging out by itself. We call molecules with such lone single electrons radicals. And they’re lonely so they can go out on a search for a friend. They’re looking for someone that has a lot of electrons that are “vulnerable” and one place they can find them are carbon-carbon double bonds, which they can find in the monomers we use. But that molecule, if it wasn’t a radical before, it is now! You still have a lone electron, just in a different place. So then that one goes on the hunt. This leads to a chain reaction that causes the monomers to link up into a polymer in the PROPAGATION phase
The polymer that forms depends on the monomers that are present. Our gel solutions contain a mix of acrylamide & methylene:bisacrylamide (bis) molecules. The acrylamide molecules will link together into linear chains & the bis molecules will act as “crosslinkers” creating “branches” that link the linear chains together.
Every once in a while, instead of finding another acrylamide molecule to join with, it finds a bisacrylamide molecule. And those molecules have an “extra” latching on site, so they can form crosslinks between strands. The higher the concentration of bisacrylamide, the more likely these mixed encounters will be and the more of these encounters there are, the tighter the mesh will be.
If you think of a staggered grid (like a brick wall but where the bricks are actually holes), the acrylamide would form the long, horizontal lines & the bis would form the shorter, staggered vertical lines. Now imagine this grid projecting into multiple dimensions ⏩ 3D mesh!
But every once in a while they’ll come across another radical. And when radicals get together, the chain terminates because you now have a pair they can just share. So this leads to chain TERMINATION.
But, since free radicals are so reactive, they won’t just be hanging out. So you have to get the chain started. And we get it started with the help of a redox pair.
Remember OIL RIG: Oxidation Is Loss (of electrons) & Reduction Is Gain
In redox reactions, one molecule (the reductant) gives electrons to another molecule (the oxidant).
Earlier we looked at some reducing agents (reductants) and saw how they can be useful in maintaining a cell-like redox environment during protein purification. But oxidants can be useful in biochemistry too! (and remember – you can’t have red without ox! http://bit.ly/2Yiya50
We needed those reductants because in an oxidizing environment, crosslinks in and between proteins can be formed when 2 of the amino acid letters, cysteines, gets oxidized (lose electrons) and link together to form a disulfide bridge. And we saw how we can use reducing agents like DTT, BME, & TCEP to reduce these bridges (give them back electrons) to break those bridges. These reducing agents just break up interactions between the side chains of amino acids though, not the actual backbone, so you’re not depolymerizing – the chain stays a chain.
When we make PAGE gels, we *want* to make bridges (hence the cross-linker) but the bridges we make are stronger than the disulfide bridges in proteins. So, even if you have reducing agents in your protein preps or buffers they won’t break up the gel
But we use a redox reaction to get the chain reaction going (INITIATION), so it can keep going (PROPAGATION) and then stop (TERMINATION). The initiator is an oxidant, APS (Ammonium PerSulfate), & its (thankfully not reluctant) reductant partner TEMED. (TEtraMethylEthyleneDiamine)
APS can dissociate into free radicals if you heat it up (to ~60C) but we can get it to lose an electron a different way so that we can do the polymerizing at room temp – TEMED offers this “alternative route.” It involves a couple steps.
TEMED has a tertiary amine – this is a nitrogen bound to 3 non-hydrogens with a lone pair free to share. It uses this lone pair to link up to one of APS’s Os, breaking that O’s bond to it’s old partner, releasing SO42- (a sulfate ion). And the O’s like “oh no you didn’t!) and splits back off, but leaving one electron behind. Because of some “shuffling” this lone electron ends up on a carbon.
Now that you have free radicals, they can free radicals “attack” the acrylamide & bis molecules ➡️ now you have acrylamide & bis free radicals ⏩ the acrylamide & bis free radicals have 2 options to give their lonely e- partners:
🅰️) attack & “latch onto” another molecule, “stealing an e-” from 1 of that molecule’s bonds ➡️ makes that molecule a free radical or
🅱️) join together w/another free radical (win-win)
Either option elongates the ⛓ but 🅰️ (propagation) keeps the reaction going 🔄 while 🅱️ (coupling) is a “dead end.” (termination)
You want the thing that the radical attacks to be the thing you want it to attack. And one thing you don’t want it to attack is oxygen, which will take that free electron, making it a radical, but not a very good initiator.
So you want to keep bubbles out of your gel. For these “quick & dirty” mini-gels, I’m not too worried about bubbles, but for larger &/or more important gels, you may want to take additional steps before adding APS & TEMED to prevent bubbles 👉
🅰️) you can bring the solution to room temp (oxygen is ⬇️ soluble in warmer solutions because the oxygen molecules have ⬆️ energy to “break free” of their interactions w/the water molecules ➡️ escape into the atmosphere)
🅱️) you can “de-gas” (aka “evacuate”) the solution by stirring it with a magnetic stir plate while under vacuum
you also don’t want bubbles in your gel because they’ll physically interfere w/the current & sample flow ⏩ inconsistent flow rates between lanes &/or crooked flow (going back to our track race analogy, a bubble would be like a hurdle placed in one racer’s lane but not the others ➡️“unfair” race ⏩ can’t accurately compare the racers)
Because APS is very unstable & continuously breaks down once dissolved in water, you want to use fresh stocks & store it in the fridge. TEMED, on the other hand, though smelly, is stable fairly long-term at room temp (but keep it tightly sealed to prevent moisture absorption & oxidation (it needs to save its oxidizing power for APS!)).
One of my favorite parts of pouring gels is when the excess gel hardens around the pipet tip (something I watch for to help know when the gel’s set) and it’s like a magic wand…
And speaking of setting, it happens fairly quickly, so you want to add the initiators right before you’re ready to pipet them in the glass casing that’ll make them set in the right overall shape.
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0