wujien wrote:
> I'm isolating a signal molecule from a fungus' culture. The MW of the
> molecule should be no more than 1000 Da. The molecule is water soluble
> and can not be extracted by organic solvent. It also can not binding
> with ion-exchanged resins (except strong acid ones; however, it is
> sensitive to strong base). I have to use sephadex G10 and G15.
> Unfortunate, it is seemed that the molecule binding with the sephadex
> (activity lost >90%). Who can give me some advices to resolve this
> problem.
With the available data one can only speculate.
Since the molecule does not interact with ion exchange resins, it is not
a phosphorylated compound (PIP3, nucleotides or the like) and not a
cation like Ca. Interaction with strong cation exchangers would point to
a week base, may be an amine.
Lipid metabolites like DAG would be extracted by solvent, as would be
unmodified steroids (but not glycosylated ones).
Your data do not exclude a peptide (possibly even a cyclic one), 1000 Da
would be about 9 AA.
Purely hypothetically it might also be an oligosaccharide, in which case
borate-chromatography could be useful.
Note that you could use negative purification: Ultrafiltration to remove
large molecules, ion exchange (either mixed bed or cationic and anionic
successively) resigns to remove charged species, and solvent extraction
for the removal of lipophilic compounds. At that stage it might be worth
checking the mass spectrum, just to see how many different compounds
remain.
Further purification could be achieved by reversed phase chromatography.
Binding to Sephadex is sometimes observed with moderately hydrophobic
compounds like some dyes (e.g. bromophenol blue) and a big pain in the
neck, because you need huge volumes of eluents to get the stuff off the
column again. You could try eluting with ethanol or propanol / water
mixtures.
Better results than with unmodified sepharose may be obtainable with
LH20-Sepharose. Pharmacia used to have a booklet on that stuff, but IIRC
it has been out of print for several years now. Check with some older
collegues.
