The Basics: How Phenol Extraction Works

by on 12th of February, 2008 in Nucleic Acid Purification and Analysis

Phenol extraction is a commonly used method for removing proteins from a DNA sample, e.g. to remove proteins from cell lysate during genomic DNA preparation. It’s commonly used, but not commonly understood.

If you want to know how it works so you can show off to all of your friends… read on.

The basic protocol

I’ll start with a quick outline of how the procedure is performed. First, a volume of phenol is added to the aqueous soup containing the proteins and the DNA to be purified.

Since phenol and water are immiscible, two phases form – a water (a.k.a. aqueous) phase and a phenol phase. Phenol is the more dense of the two liquids so it sits on the bottom.

The phases are then mixed thoroughly. This forces the phenol into the water layer where it forms an emulsion of droplets throughout. The proteins in the water phase are denatured and partition into the phenol, while the DNA stays in the water.

The mixture is then centrifuged and the phases separate. The DNA-containing water phase can now be pipetted off, and the phenol/protein solution is discarded. Commonly, the DNA is then de-salted and concentrated using ethanol precipitation.

First, a bit about solvents

To explain how the addition of phenol can separate DNA and proteins, we need to briefly touch on solvents. This is the chemistry bit… bear with me.

phenol-water-polarity.gifA solvent is a substance, normally a liquid, that can dissolve other substances. Broadly, solvents can be classified according to their polarity, which depends on how extreme the spread of the electron density in the molecule is.

Water is a very polar solvent because the oxygen atom is very electronegative so it “sucks” the electrons towards it and away from the hydrogens, creating a slight negative charge on the oxygen and a slight positive on the hydrogens. i.e. the charge is “polarised” within the molecule.

Phenol is a less polar molecule than water. Although it has a highly electronegative oxygen, this is counteracted by the phenyl ring, which is also very electronegative so there is no concentration of electron density around the oxygen. i.e. the charge is not so polarised in a phenol molecule.

DNA is most soluble in the water phase

So what does this have to do with the separation of DNA and protein?

Well in general, polar (charged) compounds dissolve best in polar solvents and non-polar molecules dissolve best in less polar or non-polar solvents.

DNA is a polar molecule due to the negative charges on it’s phosphate backbone, so it is very soluble in water and less so in phenol. This means that when the water(+DNA +protein) and phenol are mixed in the protocol, the DNA does not dissolve in the phenol, but remains in the water phase.

phenol-ext1.gifThe solubility of the proteins is flipped by phenol

But, proteins are a different story entirely.

As you know proteins are made up of long chains of amino acids. Each amino acid has it’s own characteristics, due to the nature of their side chains. Some, (e.g. phenylalanine, leucine, and tryptophan) are non-polar, because their side chains contain no charged entities. Conversely, amino acids with side chains containing charged entities (e.g. glutamate, lysine and histidine) are polar.

The polarity differences in the side chains are biologically important because they largely determine how peptides fold into functional proteins. Put simply, the chains fold so that as many as possible of the side chains that are less polar than the solvent are on the inside of the proteins (away from the solvent), while those that are of similar polarity to the solvent are arranged on the outside of the proteins (see panel 1 in the figure above). Another way to think about it is that polar side chains are hydrophilic, and non-polar are hydrophobic. The hydrophobic side chains hide on the inside, with the hydrophilic chains on the outside.

In the cell (and note, I am talking about cytoplasmic proteins here), the proteins are folded according to the influence of water as the solvent, but when the proteins are exposed to a less polar solvent, like phenol, their folding changes (see panel 2 in the figure).

Basically, the proteins flip inside-out. The less-polar residues, which hid inside the protein structures in water, now want to interact with the less-polar phenol so are forced to the outside. Conversely, some of the very polar residues may flip to the inside of the globular protein to be shielded from the unsuitable new solvent.

In short the proteins are permanently denatured by the new solvent environment provided by the phenol.

Whereas in water the polar residues on on the outside of the proteins made them soluble in water, the phenol-induced folding changes forced the phenol-favoring residues to outside so that the proteins are now more more soluble in phenol than in water.

And this is the basis of the separation. The phenol-soluble proteins partition to the phenol phase while, as discussed above, the water soluble, polar DNA molecules stay in the water phase (see panel 3 in the figure).

So that’s how phenol extraction works. If you have any questions, or corrections, be sure to let me know.

28 thoughts on “The Basics: How Phenol Extraction Works”

  1. such.ire says:

    The partitioning of proteins depends on the relative energetics of the phenol solvation of hydrophobic groups versus the folding and hydration energetics in aqueous phase. A priori, there is no reason to think that proteins wouldn’t just stay folded in the aqueous phase rather than unfolding and moving into the organic phase.

    And why phenol, as opposed to, say, methylene chloride, or chloroform? Because it’s more environmentally friendly or something?

  2. Nick Oswald says:

    Thanks for your input. Here’s how I think this works – let me know what you think.

    I think that the key is that the denaturation/re-arrangement of the proteins in phenol is irreversible. This drives the reaction in favor of the phenol-favored folding arrangement

    The proteins don’t unfold then move into the organic phase – they move into the organic phase then unfold.

    So if the protein is very water soluble, there will always be at least some dissolution in the organic phase, which will allow the process to start.

    As for solvent choice. Chloroform is normally used along with the phenol, apparently to give a clearer separation.

    But I am not sure why this combination was chosen, although I doubt the motivations were environmental – when this protocol was invented, few people really thought about the environment! :)

  3. such.ire says:

    Hmm, so, you think it’s a kinetic rather than an equilibrium partitioning, in that the protein just gets trapped in its denatured, phenol-solvated form? It seems like a lot of proteins renature pretty quickly under most aqueous conditions, so proteins would equilibrate rather quickly, leading me to think that the extraction is an equilibrium procedure. It’s not like we do phenol extractions in the cold room.

    That alcohol group is quite acidic (pKa 9.95); maybe it is somehow contributing to the solubilization of the protein? Amide bonds are pretty basic, especially on the carbonyl oxygen, so I’d expect that acids work pretty well to solubilize proteins. Perhaps it’s a combination of the packing of phenols around the amide moiety and the solubilization of hydrophobic side chains by the aromatic rings? I think the PCI is maintained at pH 8 in order to make sure that RNA and DNA aren’t getting protonated and extracted as well.

    I think I’ve found a good explanation hinting at the reason why one uses chloroform along with the phenol: not only to make the solutions less miscible, but also to force the organics to the bottom layer, for the ease of handling the aqueous layer. Apparently phenol-water just borders on the same specific gravity as salt-heavy water, which can cause phase inversion. PCI, on the other hand, won’t ever invert under normal circumstances.

    This website seems to be a pretty good resource on phenol extractions:
    http://lclane.net/text/phenol.html

  4. Nick Oswald says:

    Well…. I’ve been thinking about this a lot and you could be right. I’m going to look into it a bit further – it may be that the article needs to be re-written! Thanks for your input

  5. Les Lane says:

    Phenol is an acid (pKa=10) which is stronger than water (pKa=13). The amide group is a base (pKa=-2). Hydrogen bonding of phenol to the peptide backbone is almost certainly responsible protein solubility. Otherwise cyclohexanol would be a reasonable protein solvent.

  6. Kavi says:

    Hello, My question is whether PEHNOL IS USED TO REMOVE PROTEINS FROM AQUEOUS DNA? or just DNA sample?

  7. wanie says:

    here u’ve discussed about how phenol remove protein. may i know how protein adsorp phenols? because, as we know phenol is one of the dangerous an d poisonous compound, right?

  8. Matan says:

    Hi,

    As I gather, according to you’re explanation the reason for vigorous shaking of phenol and the DNA sample is to cause as much interaction between the phenol and aqueous phase, to form good emulsification. In my experience with phenol extraction – yield of DNA (ug/ul) increases significantly if the shaking is done very vigorously. How would you explain this, if the DNA constantly stays in the water? according to your article one should be able to mix the two phases gently, centrifuge, and collect the aqueous phase with the DNA with no loss in efficiency, since the DNA never left the water.

  9. Kuba says:

    Thanks for the article, finally a specific answer to the questions “how we denaturate protein without touching the native state of nucleic acids”;)

    Matan – what is the problem? Yield of DNA (ug/ul) increases significantly if the shaking is done very vigorously BECAUSE DNA stays in the water. Why it stays in the water? Because it’s not constructed with particulary hydrophobic “parts” and even if – the hydrophobic/hydrophilic(phosphate backbone) ratio stays equal along whole DNA molecule, in opposite to protein chain where hydrophobic aminoacids can be set just by the hydrophilic aminoac. region.
    Is that it?

  10. kang xu says:

    I still have 3 questions you did not address to:
    (1) Since phenol phase co-exist with water phase, is it possible that some protein still remain in the water phase by maintaining its orginal fold? In this manner the DNA will still co-exist with protein.
    (2) Protein is dissolved in phenol after “turning inside out”, that’s right. but centrifuge cannot separate a solvate from its solvent as long as it’s real “solution”. Only “suspension” can be broken up by centrifugation. Right?
    (3) Does phenol cleave peptide bond?
    Thanks,

  11. sapinder bali says:

    hey….one simple question..I have been facing the problem of phase inversion during phenol extraction….my DNA containing phase seeps under the phenol layer….Can you tell me why it happens??

  12. antiant says:

    Hey,

    Thanks for posting this. Really helped me out!

  13. Victoria says:

    This was an excellent and very well-written article. I’m in the last year of my microbiology/biochemistry undergraduate and sometimes it’s hard to find well-written articles which explain all of the science in an easy-to-read-matter. I’ll definitely come back to this site for other articles like this.

  14. Brian Cady says:

    So does the protein migrate across the phenol:water border by diffusion, leaving some protein behind with the DNA in the water? Or does the protein flee the water completely, due to hydrophobicity of some of the amino acids?

    I think the former, but want to check my understanding.

    Brian

  15. Y. von Grabowiecki says:

    Nick:”[...]As for solvent choice. Chloroform is normally used along with the phenol, apparently to give a clearer separation.
    But I am not sure why this combination was chosen,[...]”

    From what i know, phenol is a little soluble in water… Which not only falses your concentration readings but can inhibit enzymatic reactions and so on.
    Chlorophorm however mixes well with phenol and doesn’t mix (at all?) with water (less than phenol does). Thus chlorophorm essentially draws phenol away from water and your nucleic acids.

  16. toolika says:

    so,this flip on mechanism in a different solvent is permanent or it restores the previous structure in water?

  17. Jode says:

    sapinder bali- This is likely just a density issue. You aqueous phase is probably ‘weighted down’ with a high concentration of salt or some other solute. Normally, if you dilute the aqueous phase a bit and spin again, the organic phase returns to the bottom.

  18. sujitha says:

    hi ,
    What happens when protein is precipitated with phenol means what is the exact principle behind?
    Does phenol denatures protein?

  19. isha says:

    Hi,
    As u’v said that polar side chains are present outside the protein and less polar are inside….so how can the protein dissolve in phenol, a less polar solvent when it is already present in aqueous solution

  20. Avatar of allison allison says:

    thank you!!

    This article and the ethanol precipitation article was excellent. I went through so many other sources that never explained the theory behind the procedure. This was a huge help for my biology assignment

    Do you have an article on denaturing high performance liquid chromatography (DHPLC)?

    thanks!

  21. Avatar of yoobios yoobios says:

    Thank you
    I really love this website ^ ^

  22. Avatar of steffi steffi says:

    Hello Nick,
    I’m perplexed with some of the steps of the “acidic phenol” RNA extraction technique and was wondering whether you could please answer my following questions.

    1)I can’t really understand how phenol inhibits the function of an RNAse. Does it bind to RNAse’s active site, or maybe causes an allosteric change to the enzyme?

    2)Why after centrifugation with RNA,DNA and acidic phenol, does DNA go to the “organic phenol phase” but the RNA goes to the ”aquatic phase”? An important fact to remark here is that both are single-stranded after ”Vortexing” the solution in a previous step. So,how do these single-stranded macromolecules separate since they only differ in one hydroxyl group in the ribose?

    3)Does phenol move to the lower area of the tube because it has a bigger Molecular Weight than say any of the molecules of the NETS solution? (in the first centrifugation when separating RNA from DNA, RNA goes to the upper “aquatic phase” together with NETS)

    4)Also, I’ve read that in the step of separating RNA from the surrounding proteins we centrifuge with chloroform and neutrally charged phenol. And that chloroform ”forces-pulls” phenol to move towards the organic phase. What does this mean in molecular terms? Is chloroform a polar molecule that interacts non-covalently with polar phenol?

    5)Finally, something else that I cannot seem to find anywhere is how does lithium chloride cause RNA to precipitate but not DNA.

    Thank you very much

    1. Avatar of Jim J. Jim J. says:

      Steffi-
      RNase is an enzyme and most enzymes are composed primarily of proteins. The RNAase enzyme is no exception and it’s amino acids must be in a specific physical conformation in order to maintain functionality (chewing up RNA). The phenol essentially flips the amino acid residues inside-out, shifting the RNase out of it’s functional (native) conformation, rendering it useless.

      I’m going to take a stab at your second question as well and I hope I will be corrected if I am mistaken. Both RNA and DNA dissolve in the water (aqueous) phase because both molecules have negatively charged backbones. This is why RNA purification protocols include a step where DNAase is allowed to chew up the DNA in the sample (and vice versa with RNAase in DNA isolations).

      If I understand your third question correctly the answer is phenol is the denser of the two liquids.

      The author of this article has another great article on ethanol precipitation of nucleic acids that could shed some light on your LiCl question.

  23. Avatar of martink martink says:

    I have a question. When i do the phenol:chloroform extraction fo RNA, after the centrifuging the aqueous phase is still yellow and it smells like phenol. This does not happen with all the samples, just with some – they are samples of plant roots.
    Does any of you have similar problems? What woud you sugest to do? another shaking and centrifuging?

    Thanks for your answers!
    Martin

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  25. How does gentle shaking denature protein and transfer them to phenol?
    SDS mediated denaturing is important step before phenol extraction.
    Such uncoiled protein can be transferred to phenol and detached SDS may remain in water phase. amphipathic SDS may have important role to play at phenol/water boundry

  26. Avatar of Emma Emma says:

    Hi everyone,

    I have been reading this article with interest. At the moment I use phenol in my DNA extraction method with SDS and sodium phosphate. I am looking at the feasibility of phasing out the phenol for something less toxic and therefore easier to transport. I gather it needs to be something ideally that is less polar than water like phenol, but I’m not sure of what less hazardous chemicals may do the same job.

    Does anyone have any suggestions? Thanks in advance.

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