Denaturing vs. reducing - especially in the context of gel electrophoresis, such as SDS-PAGE
Don’t confuse “denature” with “reduce” - only a reducing agent will set cysteines loose!
blog form: https://bit.ly/denaturing_vs_reducing
note: short text with links to more detailed posts
Denaturing refers to “unfolding” and it only affects weak, non-covalent, interactions (charge or partial-charge based attractions as opposed to hard-core bonds where electrons are actually shared). It’s like untangling a charm bracelet. Common denaturing agents include heat, SDS (often used for proteins), urea & formamide (often used for nucleic acids).
But what if a couple of the charms are glued together? This would prevent you from fully untangling it. Or if charms from different bracelets we’re glued together, they’d still be stuck together no matter how hard you tried to untangle them. These are like disulfide bonds that make cystine crosslinks between or inside proteins. They’re resistant to plain-old “denaturing.”
But now imagine you bring in some glue remover. Now you could separate those stuck charms and therefore fully unfold things and separate the linked bracelets. This is equivalent to reducing. Reducing agents (such as BME, DTT, TCEP, & glutathione) break up disulfide bonds that make cystine crosslinks between or inside proteins.
And, importantly, neither of these is really “degrading” things in the sense that they’re keeping the chains in tact, not separating them into individual chainlinks. Even your glue-be-gone can’t mess with those stronger metal linkages. Similarly, “normal” covalent bonds are resistant to being broken up even by reducing agents. For reasons I explain in my cysteine & redox posts, although they are “covalent” (involve the sharing of electrons), disulfide bonds are weaker than the carbon-carbon, carbon-oxygen, etc. covalent bonds making up the molecules.
So, your chains are left chain-y unless you make conditions really harsh such as by adding a strong acid or base, or by adding a molecule-specific-chewer like an RNase, DNase, or protease.
When we run gel electrophoresis to separate molecules by size, we can run them under non-denaturing (aka “native”) or denaturing conditions. This applies to nucleic acids (e.g. plain TBE vs TBE-urea PAGE, often w/formamide in the loading buffer) as well as proteins (native vs. SDS-PAGE).
In the case of proteins, we have the extra option of reducing vs. non-reducing. We typically use reducing conditions because we want to get the molecules totally unwound and individual so that we can separate them by their linear length (based on # of amino acids) and not their shape or who they’re hanging out with. Reducing agents degrade over time in solution, so we typically add a reducing agent such as BME to our loading buffer “fresh” or at least store our loading buffer in the freezer once we’ve added it.
For preparing gel samples, denaturing agents usually act with the help of heat. So you add a loading buffer containing them to your sample, heat them up, and then send the samples on their journey. To make sure they don’t refold, you include denaturant in the running buffer and/or gel (SDS in SDS-PAGE running buffer and urea in urea-PAGE gels).
a couple notes:
note 1: I’ve been talking about reducing in the context of gels, but we also use reducing agents a lot in the lab, in the (hopeful) absence of denaturation. Because intracellular conditions are a reducing environment (with cells stocking up on glutathione as a reducing agent), a lot of the proteins we study are used to living in a reducing environment, so we make them feel comfortable (and prevent spurious cross-link formation) by adding a reducing agent to most of the buffers (pH-stabilized salt waters) we use.
note 2: “Reducing” doesn’t just apply to breaking of cystine crosslinks. In general, it refers to “loss of electrons or electron density” - I’m not going to go into it here but check out that redox post if you want to know more.
more on SDS-PAGE: http://bit.ly/sdspageruler & • SDS-PAGE theory & practice: into the ...
more on native-PAGE: https://bit.ly/nativepageoverview & • Variability in protein isoelectric po...
more on nucleic acid PAGE: http://bit.ly/ureapage & • Nucleic acid PAGE (Polyacrylamide Gel...
more on cysteine: http://bit.ly/cysteinecrosslinks & • Cysteine, sulfur, disulfide bonds & r...
more on redox (reduction & oxidation) & reducing agents: https://bit.ly/redoxbiochem & • Cysteine, sulfur, disulfide bonds & r...
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com
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