Jun 13, 2016
Phenol extraction of proteins
- David Dunigan and Irina Agarkova1
- 1University of Nebraska-Lincoln
- VERVE Net
External link: http://lclane.net/text/phenol.html
Protocol Citation: David Dunigan and Irina Agarkova 2016. Phenol extraction of proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.esgbebw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 29, 2016
Last Modified: February 15, 2018
Protocol Integer ID: 2600
Guidelines
Reagents
Liquid (88%) phenol
1% w/w Ficoll 400
0.5M DTT solution (77 mg/ml)
10M LiCl
10% w/v SDS
1% Ponceau S (dye)
Acetone
Purifying proteins
Proteins are easily precipitated from phenol with 10 volumes of methanol. I use Ficol l as a carrier. Ficoll stabilizes the pellet and makes it visible. Four volumes of methanol efficiently precipitates all but the smallest proteins. Acetone precipitates phospholipids. Methanol won't precipitate alcohol soluble proteins (usually of little concern). Methanol precipitates proteins poorly from acidic solutions. An organic base or a buffer (e.g. ammonium acetate ) solves this problem. Adding sucrose during protein extraction conveniently 'inverts' phases.
How efficient is phenol extraction of proteins?
The distribution coefficient of proteins into the phenol phase is greater than 100 (Pusztai, A. Biochem J 99 , 93 ; 1966, in my experience greater than 1000). Hydrogen bonding to the peptide backbone is important; small peptides likely have lower
distribution coefficients. Phenol is less a denaturant than a solvent. Salt (or higher pH) helps to extract histones and heat, higher pH or denaturants help to strip TMV capsid from the virion. Phenol (above, carbolic acid) was isolated from coal tar in 1834. It served as a bacteriocide in the late 19th century. Phenol extraction first served to purify (deproteinize) carbohydrates (Westphal, et al. Z Naturforschung B 7 , 148, 1952). It was subsequently adapted to 'purify' nucleic acids (Kirby, Biochem J 64 , 405, 1956 ). It also separates glycoproteins (Howe, et al, Meth Enzymology 28 , 236, 1972) from erythrocyte membrane non-glycoproteins.
Basic principles
Phenol dissolves proteins (for an interesting example see Cohn, E.J. and Conant, J.B. The molecular weight of proteins in phenol . PNAS 12 , 433- 438, 1926) and lipids leaving water soluble matter (carbohydrates, nucleic acids, etc.) in the aqueous layer. Particulate and 'ambiguous' matter remain insoluble. Phenol extraction of nucleoproteins (e.g virus particles) gives pure products. Crude tissue yields complex mixtures, particularly in the aqueous phase.
Preparing phenol
Phenol is often distilled and then saturated with water. Chelators (8-hydroxyquinolin e) are added to inhibit oxidation. Phenol is usually neutralized by dialyzing against a buffer. Biochemists have ignored the acidity of phenol (pK ~10) which is itself the major buffer above pH 7 (and will titrate buffers proportional to the buffer/phenol ratio). It is far simpler to neutralize phenol by adding a weak base (such as tris, I prefer N-ethylmorpholine, a liquid). Adjusting the phenol/tris ratio is more convenient (and reproducible) than dialyzing. Raising pH stimulates oxidation so neutralize phenol immediately before use.
To 100 µl of PBCV-1 in a 1.5 conical plastic centrifuge tube add 150 µl of phenol.
Add DTT (~10 µl), 10% SDS (~20 µl), 10M LiCl (~15 µl) and Ponceau S (~10 µl).
Heat 5 min at 75°C, mix thoroughly.
00:05:00
Allow to cool, centrifuge 1 min (5K).
00:01:00
Draw off lower (clear) phase into 1.5 conical plastic centrifuge tube.
Add 1 ml acetone and then 5 µl of 1% Ficoll, mix.
Store 20 min at -20°C.
00:20:00
Centrifuge 1 min (6K).
00:01:00
Discard supernatant, drain tube, wash ppt once with acetone (ppt sticks to tube) and dry.
Dissolve pellet in ~50 µl cracking buffer (1% SDS; 5% glycerol; 0.03125 M Tris-HCl, pH 6.8; bromophenol blue-trace).