Agarose Gels Do Not Polymerise!
I was browsing a certain website the other day when I came across a protocol that advised: “When agarose starts to cool, it undergoes what is known as polymerization”
Polymerising agarose? Someone is getting their gel matrices mixed up, and it’s not the first time I have heard this said, so it looks like some myth-busting is required.
So hunker down for a quick run down on the difference between polymerising and non-polymerising gel matrices.
Of the common gel matrices used in molecular biology, polyacrylamide, agar and agarose, polyacrylamide is the one that polymerises. I’ll deal with agarose and agar later.
Polyacrylamide gel polymerisation
Polyacrylamide, used mainly for SDS-PAGE, is a matrix formed from monomers of acrylamide and bis-acrylamide. It’s strengths are that is it chemically inert – so won’t interact with proteins as they pass through – and that it can easily and reproducibly be made with different pore sizes to produce gels with different separation properties.
The polymerisation reaction, shown in the diagram below, is a vinyl addition catalysed by free radicals. The reaction is initiated by TEMED, which induces free radical formation from ammonium persulphate (APS). The free radicals transfer electrons to the acrylamide/bisacrylamide monomers, radicalizing them and causing them to react with each other to form the polyacrylamide chain.
In the absence of bis-acrylamide, the acrylamide would polymerise into long strands, not a porous gel. But as the diagram shows, bis-acrylamide cross-links the acrylamide chains and this is what gives rise to the formation of the porous gel matrix. The amount of crosslinking, and therefore the pore size and consequent separation properties of the gel can be controlled by varying the ratio of acrylamide to bis-acrylamide.
For more information on polyacrylamide gel polymerisation see Biorad Bulletin 1156
Agarose gel formation
So what about agarose? Well, agarose – the main component of the gelatinous agar that can be isolated from certain species of seaweed – is itself a polymer. But, polymerisation is not the mechanism for agarose gel formation.
Chemically, agarose is a polysaccharide, whose monomeric unit is a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose which is shown in the diagram below.
In aqueous solutions below 35°C these polymer strands are held together in a porous gel structure by non-covalent interactions like hydrogen bonds and electrostatic interactions. Heating the solution breaks these non-covalent interactions and separates the strands. Then as the solution cools, these non-covalent interactions are re-established and the gel forms.
So agarose (and agar) gels form by gellation through hydrogen bonding and electrostatic interactions, not through polymerisation
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Nick Oswald
Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn
HAHA! I _hope_ you bonked the other blogger on the head! or at least left a comment. There should be a list of “Top 100 Biggest Bench Myths” somewhere. (hint hint)
But, somehow, can we define each agarose polymere strand as a “monomer”? If so, the interactions between the strands can be assumed as a “polymerisation-like” process, no ? (^_^)
Is the polymerisation process only based on covalent interactions ?
@Jonathan: â€?Top 100 Biggest Bench Mythsâ€? – that’s a good idea. But 100 is a tall order! Maybe I should put out a call for our readers to put forward their favorite myths then compile them into an article. Thanks for the suggestion!
@GG: Thats a great question (that’s my standard response to a question at the end of a presentation that I need time to think about, but it really is a good question!).
I had always assumed that polymerisation involves covalent bonding. To me, a matrix of agarose “monomers” held together by hydrogen bonding is no more a polymer than ice is a polymer of water.
If you do a Google search for “non-covalent polymer” there are references to “polymers” made of hydrogen bonded monomers. But according to a IUPAC document on definitions of terms in polymer science, a polymer is:
“A molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass.”
(http://www.old.iupac.org/reports/1996/6812jenkins/6812basicterms.pdf)
I’m not the world’s greatest at pure chemistry but the key point here is that the polymer has an increased molecular mass compared to the monomer. I think that molecular weight specifically refers to covalently or ionically bonded atoms so this definition of a polymer would exclude hydrogen bonded monomers.
Can anyone else help out here?
As a rule, you do normally need to have covalent bonding to achieve polymerisation. Certainly, in the sense of forming these sorts of gels, it’s not a polymer forming – in the hydrogen bound sense above you need repetative hydrogen bonds to really define a polymer.
If the system is somewhat ordered, then it probably counts as a polymer. If the hydrogen bonds are essentially random, then it’s not so much a polymer, and more of a condensed state.
Thanks Dr Ace
[...] that I previously wrote an article about the mechanism of acrylamide polymerisation previously – click here to read it. The discontinuous buffer system and the stacking gel – lining them up at the starting [...]
that’s why u can boil it again and get it liquid!
Well put. Although to my mind true covalent bonds, hydrogen ‘bonds’ and van der Waals dispersion type interactions are actually just variations of molecular orbitals of differing configuration with the same underlying mathematics and physical basis. But no question that they behave quite differently from a chemical perspective.
why do we use polyacrylmide gels in sds page and not agarose gels?? what makes the difference??