Drop-dialysis, help

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Aug 3 04:56:19 EST 1999


Hi Jeff,

> Does that really work?

I wouldn´t give that tip if it didn´t work.

> How can you determine that salts are removed
> during this kind of dialyis?

1. You see it when you have a high salt solution drop and you look at the floating
filter from the side: the salt "falls through" the filter.
2. salt-sensitive experiments work after, but not before dialysis.
3. You can measure the conductivity before and after dialysis (see below)

> The amount of the volume to be dialyzed
> is too small to determine its conductivity after dialysis.

No. It depends a bit on the amount of salt before dialysis, of course, and the
sensitivity of your conductivity-meter, but I use to take 3microliters, dilute
100fold to 1000fold, and determine conducitivity. It works well for me.

> BTW, what is the molecular cut-off ranges?

That is something I can´t tell. I have never found a technical data sheet that
states the cut-off. But what I can say is that a 41kDa proteins is not lost during
the procedure, nor is a tetrameric protein of 58kDa. So, the cut-off appears to be
lower than 40kda, which is sufficient for my needs.
Ok, just because you asked, I have set up a little experiment with cytochrom C
(12kDa) dissolved in TNT buffer to give a red-brown solution (I have not bothered
to weight the protein). In a parallel control, I have mixed NaCl into the solution
to 2M final conc. I used the setup I described with 50ul drops. The experiment is
running for 90min now. I didn´t see any colored protein fall through, but I did
see the salt in the second drop (only apparent in the first minutes of dialysis).

Thus, cutoff appears to be even lower that 12kDa, but well above salt (mw=58).

Frank

> I am just curious.
> jeff
>
> On Mon, 02 Aug 1999 14:03:19 +0200, "Frank O. Fackelmayer"
> <Frank.Fackelmayer at uni-konstanz.de> wrote:
>
> >Hi,
> >For dialysis of small volumes, we put a drop of the stuff (between 10 and
> >100microliters) onto the hydrophobic (shiny) side of a Millipore VS filter
> >(0.025micrometer pores), floating on 20 to 50m buffer in a plastic petri dish.
> >Let stand undisturbed for 2h in the cold, then take up the drop with a pipette
> >again.
> >
> >Hope this helps,
> >Frank




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