I am extracting Lentinan from Shiitake and the method says that I need to remove protein usin the Sevag method. I have searched and the only thing that I have found is the usage of chloroform and octanol, but I haven´t found the exact method
The exact method is simple. Take your crude lentinan extract and shake hard with 1 volume of butanol/chloroform (1/5) mixture in a 50 ml bluecap tube. Centrifuge the emulsion at 2500 rpm 10 min and take the upper layer. Interphase contains denatured protein. Precipitate the lentinan by adding 2 vol. ethanol and keep at -20 for 1 hr. Centrifugation will give a pellet of partially purified polysaccharide. Pure lentinan is white.
The exact method is simple. Take your crude lentinan extract and shake hard with 1 volume of butanol/chloroform (1/5) mixture in a 50 ml bluecap tube. Centrifuge the emulsion at 2500 rpm 10 min and take the upper layer. Interphase contains denatured protein. Precipitate the lentinan by adding 2 vol. ethanol and keep at -20 for 1 hr. Centrifugation will give a pellet of partially purified polysaccharide. Pure lentinan is white.
Sorry to intrude on the question, but Professor Van Griensven, can you further explain the step after the centrifugation of the emulsion? I'm having a hard time understanding what exactly you meant when you said to take the upper layer and then the interphase that contains the denatured protein. Is it the interphase of the upper layer or the interphase that remains in the bluecap tube after we remove the upper layer?
Interphase is the junk layer between the water layer and the chloroform layer. Denatured protein is for large part in the interphase (and might stick to the wall of the tube; take care)
The reason might be dat polysaccharides may bind (some)polyphenols irreversibly.
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