Question
Asked 22nd Nov, 2017

What is the principle of methanol:Chloroform protocol for protein precipitation?

I need to know the chemical principles in protein precipitation.

Popular Answers (1)

23rd Nov, 2017
Snigdha Maiti
St. Jude Children's Research Hospital
The use of methanol,chloroform and water protocol to precipitate the protein is used for removal of interfering agents such as lipids, nucleic acids from the protein samples which will greatly enhance the resolution of proteins on acrylamide gels. Protein precipitate forms at the interphase of a top aqueous layer(MeOH+H2O) containing salts, detergents, reducing agents, nucleic acids etc., and a bottom layer of chloroform containing lipids. By carefully removing the interphase from the aqueous phase will yield dry protein materials free of detergents and salts which is needed for LC/LC-MS etc.
This method may be used to concentrate proteins from diluted samples and may be used as a sample cleanup procedure for western blotting, regular SDS-PAGE, 2D gels, etc.
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All Answers (3)

23rd Nov, 2017
Snigdha Maiti
St. Jude Children's Research Hospital
The use of methanol,chloroform and water protocol to precipitate the protein is used for removal of interfering agents such as lipids, nucleic acids from the protein samples which will greatly enhance the resolution of proteins on acrylamide gels. Protein precipitate forms at the interphase of a top aqueous layer(MeOH+H2O) containing salts, detergents, reducing agents, nucleic acids etc., and a bottom layer of chloroform containing lipids. By carefully removing the interphase from the aqueous phase will yield dry protein materials free of detergents and salts which is needed for LC/LC-MS etc.
This method may be used to concentrate proteins from diluted samples and may be used as a sample cleanup procedure for western blotting, regular SDS-PAGE, 2D gels, etc.
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27th Nov, 2017
Alejandro Martin
Center for Genetic Engineering and Biotechnology
If you've ever deproteinized a DNA sample by extraction with buffered phenol, it is pretty similar. First, you create a two phase (MetOH and water on the top, CHCl3 on the bottom) system. Proteins, being amphiphilic, will denature to expose hydrophilic residues to the aqueous phase and hydrophobic residues to the organic phase, forming a layer at the interface. Then it's only a matter of removing the upper aqueous phase, adding MetOH to the CHCl3 phase to decrease its density, and then spinning the aggregated protein (which will no longer float on the organic phase) and collecting the pellet.
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11th Dec, 2017
Engelbert Buxbaum
Private Person
The method was described in
@ARTICLE{Wes-84,
title = {A Method for the quantitative recovery of protein in the presence of detergents and lipids},
author = {D. Wessel and U.I. Fl{\"u}gge},
journal = {Anal. Biochem.},
year = {1984},
pages = {141-143},
volume = {138},
abstract = {chloroform/methanol precipitation},
doi = {10.1016/0003-2697(84)90782-6},
language = {eng},
}
At first, the protein is denatured in a homogeneous solution of water, methanol and chloroform. Adding further water makes the chloroform precipitate out of this solution as fine droplets, with the protein at the interphase. After centrifugation protein forms a thin film between the layers. You remove hydrophilic contaminants with the aqueous, and lipophilic ones with the chloroform phase.
In addition, as you can dissolve the film in, say, 10 µl buffer, you get a 100-fold concentration of the protein.
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