Could that be a massive protein I see? We can separate proteins by size by sending them traveling through an SDS-PAGE gel, but in order to find our protein treasure we need to send in some treasure hunters. And the one we use most often is a dye called Coomassie Brilliant Blue (CBB).
You can think of the SDS-PAGE gel’s matrix as maze w/protein “treasure” hidden throughout. The dye travels randomly thru maze by diffusion (the molecules move around randomly, ricocheting off the things they run into with the NET RESULT that they move from areas of high concentration to areas of low concentration). And when it finds protein treasure, it latches on. And since the dye’s blue it tells us where the protein is.
But it’s really hard to find treasure that’s trying to escape! So we need to get the treasure to stay put – the FIXATION step uses an alcohol and/or acid to make the protein to precipitate (clump up) so it gets stuck in place so our treasure-hunting dye can find it. We also have to watch out for overly-eager treasure hunters that latch onto “fool’s gold” (stain the gel itself) causing high background. This leads to an overall treasure-hunting scheme of
GEL ELECTROPHORESIS – separate proteins by size by unfolding them, coating them in negatively-charged detergent (SDS) & using that negative charge to motivate them to travel through a polyacrylamide gel mesh towards a positive charge. The bigger (longer) proteins get tangled up more, so they travel slower and have progressed less (so higher up on gel) when you turn off the power. More here: http://bit.ly/2GZc3tG
WASH – remove free SDS, etc.
FIX – trap the treasure – the gel has to allow proteins to move when you want them to, but not too easily – Ideally they’d only move when power’s on, but molecules like to move and if they can they will – so (especially the small ones) can start wandering off even when the power’s off) So you add an alcohol and/or acid to get them to clump and get stuck in place.
STAIN – send in the treasure hunters – stick the gel in a bath of dye – the dye enters, latches onto the protein and gets stuck too (this step’s often combined w/the fixing step)
DESTAIN – call off the hunt – get the treasure-less treasure hunters to leave so you can better see where the treasure-full ones are.
There are lots of different formulations including “Classic Coomassie” which has really eager treasure-hunters that can find tiny amounts of treasure but are also fool’s-gold-happy – it’s super sensitive and eventually gives you nice crisp bands, but takes a lot of destaining to reveal them. Alternative “Colloidal Coomassie” recipes are becoming more and more common because they’re faster & more eco-friendly – these forms keep groups of treasure-hunters hanging out outside the gel and gradually send them in until all the treasure’s found and bound.
Let’s take a closer look at our treasure hunter, Coomassie Brilliant Blue (CBB). Most scientific reagents have boring (though descriptive) names, so you might wonder how Coomassie Brilliant Blue (CBB) got its name. If you’re kinda nerdy, you might look it up. And if you’re even nerdier like I am, (embrace it!) you might then tell other people about it!
“Coomassie” is actually a trademark name – owned by a company that no longer even makes it – & a town name (modern-day Kumasi, Ghana). CBB wasn’t discovered or produced there, nope – a British company thought naming their product after the capital of the Ashanti empire they recently conquered would be good business strategy. That makes me mad, so I’m going to call it CBB most of the time.
“Coomassie” was initially used to market a wide range of wool dyes, w/CBB first made in 1913 & 1st used to stain proteins in 1960s. Dye-ing to know more about the dye itself? It has a pinwheel-like chemical structure and is characterized as a triphenylmethane dye. Unlike the common name a company decided to give a product, this is an example of a functional name – it describes the chemical makeup – in this case three (tri) phenylmethane groups – phenyl is a type of resonance-stabilized ring (a ring or atoms that share electrons in a delocalized fashion that makes them good at absorbing light and thus making things look colored) – and methyl is a CH3 group.
There are 2 main forms of this “triphenylmethane” dye, R-250 & G-250. Both are blue, but R’s more “reddish” & G’s more “greenish” (although the color depends on the pH and whether and what it’s bound to which is why it can be used to measure protein concentrations in something called a “Bradford assay”). “250” was originally a purity/strength indicator. R 250’s more sensitive , but G-250 can be made into forms that produce lower background, with faster protocols are more eco-friendly recipes.
We get the G-250-based “quick stain” we use most of the time pre-made, but we make our own “Classic” R-250 stain. It’s a really simple recipe – only 4 ingredients: water, acetic acid (AcOH), methanol (MeOH), & CBB – but it takes a while to prepare… more here: http://bit.ly/2QJNwLy
But none of that really matters if the dye’s not where you want it – and only where you want it. And where we want it is stuck to our protein (which is itself stuck in our gel). And where we don’t want it is anywhere in the gel there isn’t any protein. So how does it stick to our protein but not the gel? The bumbling biochemist’s here to tell!
CBB has sulfuric acid groups that can be negative or neutral depending on pH. Under the conditions of the staining solution it has overall ➖charge (anionic), so it binds (reversibly) to ➕-charged parts of proteins (basic amino acids like Arg, Lys, & His) through electrostatic interactions (opposites attract). (Those side chains aren’t always positive, but we stain the gel in acidic conditions, where they’re more likely to be – more here: http://bit.ly/30qzHH6
CBB also binds to non-charged protein parts (especially the ring-y amino acids like Phe, Tyr, & Trp) “generically” through “Van der waals” interactions, which involve shifting around of electrons when molecules get close together. These interactions are weak but add up (they’re what allow geckos to walk up walls!). Protein with unusually high proportions of ring-y amino acids tend to stain better. An example is BSA (bovine serum albumin), which recruits twice as many treasure hunters per weight of protein than your average protein.
And speaking of weight, different proteins have different weights because they have different #s of amino acid letters. We commonly talk about protein weights in terms of “molecular weight (MW)” and units of “kiloDaltons” (kDa). BSA’s molecular weight is 66.5 kDa, meaning that 1 mole (6×10^23) of BSA molecules would weigh 66.5 kg. More on moles here: http://bit.ly/2KQLw4k
But the key thing here is that bigger proteins have “more to love” in CBB’s eyes – they offer more binding sites and thus will more BSA per protein molecule than a smaller protein. So your bands would look darker for bigger proteins than smaller proteins if you ran the same # of protein molecules of each.
CBB’s protein-binding ability also makes it a good wool dye because wool’s chock full of a protein called keratin. And that keratin-binding ability which makes it good at dyeing wool also makes it good at dying your skin – so wear gloves & avoid splashing.
CBB can also bind SDS, which can mess up results. So you want to wash your gel in water before staining to remove the SDS.
As you can hopefully see from the pics, Classic CBB initially stains your whole gel blue so you can’t see the bands until you destain it and de-blue what’s not supposed to be blue.
This is because in order to find tiny treasure you unleash a ton of treasure hunters that race in from the dye bath, where there’s a high concentration of dye, to the pores of the gel where there’s more room – and hopefully protein! But they’ll keep snooping around even if there’s no protein left to find because you have high concentrations of free dye inside and outside the gel.
But Colloidal CBB doesn’t require as much destaining (though it helps make the bands crisper) because, instead of sending in a ton of treasure hunters you then have to kick out, it has groups of hunters hang out outside the gel and only go in “as needed”
A colloid is a solution where instead of having individual molecules spread throughout, you have “clumps” of those molecules, but still evenly spread out like in a “normal” solution. And they really are “dissolved” (they don’t come “undone” when you let the solution sit)
The colloids are too big to enter the gel so you don’t “overstain.” BUT the FREE molecules can come & go from the gel as they please – and they do, just as before, except now they’re at a lower concentration. So how do you get enough to stain the protein? No problem – the free CBB population can constantly get replenished from colloidal “stock rooms”
The result? Instead of flooding the gel with dye (like the “classical” method) you supply a steady, mild flow of CBB molecules – enough to quench the proteins’ thirst, but not so much that it can stick to the gel.
So you can see the bands even without destaining. It also helps that, although we think of CBB as blue, it’s color depends on its charge. At low pH, CBB G-250’s brownish, but when it binds proteins, it gets stabilized in blue state so you can tell background brown from treasure blue.
And speaking of color, a great thing about coomassie stains is that, unlike the DNA gels we looked at a couple of weeks ago, that used fluorescent dyes we had to stick on a UV tray, http://bit.ly/33RznDA CBB is “colorimetric” – the readout is color, so the results are literally right there to see, no special equipment required (unless you count the eye, which is a pretty spectacular tool!) (and the light tray helps too 🙂 )
This post is part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology. Be sure to follow the IUBMB if you’re interested in biochemistry! They’re a really great international organization for biochemistry.