Spin columns - revisited
akl at prophet.pharm.pitt.edu
Tue Jun 8 21:04:07 EST 1993
In article <6JUN93.23420939 at msdisk.wustl.edu>, wetsel_r at msdisk.wustl.edu
> We are looking for recipies for making one's own spin columns. ANY recipies
> would be appreciated. We need to spin our DNA after Taq cycle sequencing
> for the autosequencer to remove all the dye-terminators. Normally, we use
> the BMB G-50's but we need to streamline the "cost of materials" use and
> the spin columns are one place to do that.
> Any suggestions would be appreciated.
> Thanks in advance.
Hi everybody interested in gel filtration in centrifuge!
We are using spincolumns for over 10 years already. We are using all kinds
Bio-gel P (P-2, P-4, P-6 etc); Bio-Gel A (0.5M, 1.5M, 5M); Sepharose CL4B;
Sephadex G25, G50. It is flexible; you can purify your DNA but we mostly
work with proteins, polymers and liposomes. Obviously, on Sephadex and
you can desalt your proteins and purify them after radioiodination. On
A5M or Sepharose CL4B you can purify liposomes (maybe viruses too, :-) ?)
from unbound proteins (V.P.Torchilin, A.L.Klibanov, V.N.Smirnov.
Phosphatidylinositol may serve as the hydrophobic anchor for immobilization
of proteins on liposome surface.- FEBS Lett.,1982, 138, 117-120).
Using A1.5M spin-column Cullis recently discovered proteins which bind
to liposomes in plasma(Chonn A. Semple SC. Cullis PR. Separation of
liposomes from blood components by a spin column procedure: towards
identifying plasma proteins which mediate liposome clearance in vivo.
BBA 1070(1):215-22, 1991)
We always use disposable plastic syringes (1,2,3,5 ml) to purify
50 ul to 0.5 ml of our samples. Good thing about this method is that you
can have as many gel filtrations at a time as your bucket rotor will
usually it means at least 4.
Before filling the syringe with the gel slurry, instead of glass wool a
or porous polyethylene plug (like Pharmacia PD-10 column has) we just
into the syringe a single glass bead (3mm diameter; S/P Baxter or Fisher
have these). It fits perfectly on top of the syringe luer output; liquid
but gel granules cannot (we use large and medium grade gels which are quite
Differing from Maniatis bible, we use the protocol close to Sephadex
procedure of 1978 (Fry DW. White JC. Goldman ID. Rapid separation of
molecular weight solutes from liposomes without dilution. Analytical
Biochem. 90(2):809-15, 1978) . In this case only two spins are
required: one to dry the gel and another (exactly the same time and speed
the first one!!) after sample application. We do not use multiple
for filling the column; we use smaller speeds, too. Sometimes
400 g for 6 min is just fine. Unfortunately some gels in our hands were
too soft to be used in this procedure even at low speed (Sephadex G75 and
Bio-Gel A15 and above). Some gels adsorb some materials; e.g. Sephadex
positively charged polymers. If you want to be sure, just take some
substances and spin. Blue dextran separation from calcein or
be monitored visually, so you can tell whether all large molecules are out
gel after the final spin, and how high is filtrate contamination with low
molecular weight dye (usually you just see the dye on top of the gel, maybe
1-2 cm inside the gel).
So, good luck with your spin. Sasha.
Standard disclaimers always apply.
> haviland at kids.wustl.edu
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