> Dear all,
> I did protein extraction from fruit (Curculigo latifolia) according to the
> attached protocol. I got the white pellet. but while i left it to air dry
> briefly, i saw the color of pellet got darker, in some parts, it changed to
> black. i think while i took the phenol phase, unwanted substances entered.Do
> you have any idea of the type of the substance that causes this,
> then the pellet was solubilised with urea, thiourea, chaps. agitated for
> two hours at 4 degree then no parts of pellet remained and all solubilised.
> then i centrifuge at 12000g for 10 min, in one tube a white substance and in
> another one an almost dark substance precipitated. i took the supernatant
> for bradford protein assay.
> i would be grateful if you could help me identify these unwanted substances.
I am sure that what is causing your problem (darkening to black) is
residues of polyphenolics, biphenolics, etc. Most land plants have enzymes
called polyphenol/biphenol oxidases which cause huge problems when
attempting to purify RNA or DNA (and apparently protein). I have had a
moderate amount of experience in isolating (or trying to isolate) usable
nucleic acids from ferns, mosses, a liverwort as well as gymnosperms and
angiosperms. Generally, flowering plants that are food sources tend to be
low in polyphenols so they are usually easier to work with. Curculigo
latifolia (Molineria latifolia) is known for its protein curculin (a
potential artificial sweetener) but I would suspect the fruit does contain
lots of polysaccharides as well as apparently the phenolic compounds.
I think you have a challenging problem here. I know how nucleic acid
extraction could probably be accomplished but what you want (the protein)
is normally in the part you throw out in nucleic acid extractions, and
phenolic compounds are also normally in the organic phase except for those
permanently cross-linked to the RNA or DNA that you may be trying to
Certainly keeping a reducing agent like mercaptoethanol or DTT in the
solutions will inhibit the phenol oxidases, but I think the whole protocol
will have to be rethought to attempt to separate the protein fraction from
the phenolics as early as possible.
Your difficulties are certainly not unexpected or unique because they are
faced frequently by anyone trying to do much molecular biology on plants.