I see bubbles! Bubbles from the wires of a gel electrophoresis module are like the biochemist’s version of looking to a waving flag to know it’s windy. The first thing I do when starting a gel electrophoresis run to separate molecules by size is look for them. Unlike bubbles inside our gel itself, these bubbles, coming off from the wires are a good thing! They tell me water’s being split (electrolysis of water) and this indicates that an electric field is being formed to motivate my proteins through the gel.
disclaimer – I am NOT a physicist – I’m a biochemistry whitticist so I’m gonna try my best but you’ve been forewarned! I’m going to explain it for SDS-PAGE, which is is a technique we use to separate proteins by size, but the same concept’s going on with agarose gel electrophoresis used for separating DNA fragments by size – that just uses a thicker gel in a horizontal format. more on the similarities and differences: http://bit.ly/2Ik1g0s
ELECTROLYSIS – What’s that❓-lysis 👉 splitting & electro- 👉 done by electricity. And electricity is the movement of electrons. And when molecules transfer electrons we call it oxidation (taking electrons) and reduction (giving electrons).
to help us remember this we have a helpful mnemonic: OIL RIG: Oxidation Is Loss (of electrons (e-)); Reduction Is Gain of electrons
And to help us remember what happens where, we have another mnemonic: RED CAT; AN OX: REduction occurs at the CAThode
Sometimes the electron pass-of is direct from the initial giver to the final taker. But if you make the electrons travel to get to their final destination you make a current. And generate an electric field between the negative place electrons are fleeing from & the positive place they’re fleeing to. And, if we coat our proteins with negatively-charged SDS detergent, they’ll flee the negativity too.
But that field will neutralize itself if you let it because, unless you’re replenishing the source of electrons you are bringing the negative to the positive & negative + positive = neutral. And that’s where the power source comes in. The electrodes in the electrode assembly (running module) connect to wires in the lid of the tank ➡️ connect to the power source.
the running module has 2 electrodes:
⚫️ ➖ electrode (called the cathode) 👉 connected to🔝 of gel (through buffer in the upper (inner) chamber) 🔴 ➕ electrode (called the anode)👉 connected to ⬇️ of gel (through buffer in the lower (outer) chamber)
⚠️ If you’re used to thinking of battery terminals, you might be confused because in batteries the names of the + & – parts are are the other way around. The reason for the difference is that the electrophoresis system is NOT a battery – it’s basically the opposite. A battery is a “galvanic cell” 👉 it uses chemical rxns to produce electricity. Our system is an “electrolytic cell” 👉 it uses electricity to drive chemical rxns.
So you need to think about 2 connected electrical circuits 👉 the battery in the power source “charges” the electrodes in our electrophoresis system, so the electrode names are reversed.
In our system:
⚫️ power source adds electrons (reduces) the cathode ⏩ makes it ➖
🔴 power source removes electrons (oxidizes) the anode ⏩ makes it ➕
So to understand why battery anodes are the end, you kinda have to think of things from a battery’s point of view 👉 at its anode, oxidation means the chemicals are losing electrons, but these electrons are being “collected” to use later, so the electrode becomes ➖.
But, in our electrolytic cell, the electrons that are lost (which is still oxidation and still happens at the anode) are “shipped out” so the the anode is +.
In a galvanic cell like a battery, the electrons come from the anode actually making them through a spontaneous oxidation reaction. then those electrons travel towards the cathode, reducing the cathode. In an electrolytic cell, the electrons come from the power box and go from the power box to the cathode. So the cathode stays constantly negative and will push away as much of that negativity as it can towards the positively charged anode. And our proteins go along for the ride!
Another thing that can get kinda confusing is that Red cats live on the dark side of the electrophoresis moon! Reduction happens at the Cathode, but that cathode is color-coded black – NOT RED! Instead, An Ox is the red-colored one.
so, quick “cheat sheet” then let’s look closer at those bubbles!
black – cathode – close to wells – negative
red – anode – close to end of gel – positive
Now – to the bubbles and beyond! The bubbles are hydrogen (H2) & oxygen (O2) gases. And they get formed through redox reactions. hydrogen gas is given off at the cathode & oxygen gas at the anode
Oxidation & reduction come in pairs 👉 if something’s getting oxidized, something else must be getting reduced & vice versa 👉 oxidation & reduction are “1/2-rxns” of an overall “redox” rxn
Here, our overall redox rxn is: 2 H2O ➡️ 2 H2 + O2 (2 molecules of water are split into 2 molecules of H gas & 2 molecules of O gas)
And our 1/2 rxns are:
▪️cathode: power box sends e- to cathode ⏩ water “gains” e- (is reduced) 👉 4H2O + 4e- ➡️ 4H2 + 4OH-
🔺anode: power box removes e- from anode ⏩ water “gives 🔙/loses” e- (is oxidized) 👉 4 H2O ➡️ O2 + 2H2O + 4e-
As you know if you’ve ever opened a soda bottle, gases like to ⬆️ in liquids, & anode’s at ⬇️ of tank, so O2 it releases floats ⬆️ as bubbles (pockets of trapped gas).
Electrons flow out of the black pole (cathode) towards the positively charged red pole (anode) which has fewer electrons. But they travel there piggybacking an OH -> OH- The cathode’s negative because it’s receiving electrons from the power source. And using those electrons to split (lyse) water. And this produces hydrogen gas
4H2O + 4e- -> 4H2 + 4OH-
Even though it’s using those electrons, it keeps getting a steady supply of fresh electrons from the power source, so it stays negative. And the OH-, being negative, don’t want to hang out near all that negativity, so they travel towards the positively-charged anode (with your proteins going along for the ride!)
Molecules would rather be neutral than charged (charge makes them less stable), so when the OH-s they get there they team up to form oxygen gas & water.
4OH- -> O2 + 2H2O + 4e-
As you can see, when they team up they no longer need those electrons – they needed them before to fulfill their electronic desires, but now they get those desires fulfilled by sharing so the extra electrons get released. And then the power source steals them, so the anode stays positive. So the cathode keeps sending negative stuff their way.
The hydroxide (with it’s electrons) aren’t the only things attracted to the positive charge – the naturally negative DNA or RNA or the SDS-coated proteins are too. So they move towards the anode too. But they have a harder time than the OH- because they’re bigger & get tangled up in the mesh as they go. The bigger they are the more tangled they’ll get & thus the slower they’ll move. And this is why they get separated by size
You’re producing gas at both anodes, but it’s more obvious at the cathode because for each water molecule you get 2 molecules of hydrogen gas but you only get 1 molecule of oxygen gas at the anode.Since you have twice as many hydrogen gas molecules coming off, you’ll get twice the bubbles. So when you’re running an agarose gel, the bubbles nearest the wells will be more obvious. It’s more deceptive with SDS-PAGE gels because the bubbles are most clearly coming from the bottom. But that’s just because of how the wiring’s set up – you can see that the top wire where all the bubbles are coming from is actually hooked up to the cathode.
This isn’t the only place in SDS-PAGE where we see redox play a role. The other day we looked at how we can use reducing agents like beta-mercaptoethanol (BME) which, instead of splitting water, donate electrons to split up disulfide bridges that help hold some proteins together. We put these in our sample loading buffer to reduce these bonds which can prevent the SDS & heat from fully denaturing (unfolding) the proteins. (we need them all unfolded to even out the playing field so length of the amino acid chains, not their folded up shape determines how fast they move). http://bit.ly/2Yiya50
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0