Spin columns - revisited

Alexander L.Klibanov 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
wrote:
> 
> Greetings:
> 
> 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
of gels:
Bio-gel P (P-2, P-4, P-6 etc); Bio-Gel A (0.5M, 1.5M, 5M); Sepharose CL4B;
CL6B.
 Sephadex G25, G50.   It is flexible; you can purify your DNA but we mostly
work with proteins, polymers and liposomes. Obviously, on Sephadex and
Bio-Gel P
 you can desalt your proteins and purify them after radioiodination. On
Bio-Gel
 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
large unilamellar
 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
allow;
 usually it means at least 4.
	 Before filling the syringe with the gel slurry, instead of glass wool a
la Maniatis
 or porous polyethylene plug (like Pharmacia PD-10 column has) we just
throw 
into the syringe a single glass bead (3mm diameter;  S/P Baxter or Fisher
should
 have these). It  fits perfectly on top of the syringe luer output; liquid
can pass 
but gel granules cannot (we use large and medium grade gels which are quite
rigid).
	 Differing from Maniatis bible, we use the  protocol close to Sephadex
G-50
 procedure  of 1978 (Fry DW.  White JC.  Goldman ID.  Rapid separation of
low 
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
as 
the first one!!) after sample application. We do not use multiple
centrifugation
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
above;
  Bio-Gel A15 and above). Some gels adsorb some materials; e.g. Sephadex
binds
positively charged polymers. If you want to be sure, just take some
dye-labeled
substances and spin. Blue dextran  separation from calcein or
sulforhodamine can
be monitored visually, so you can tell whether all large molecules are out
of the
 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.
> David
> haviland at kids.wustl.edu



More information about the Methods mailing list