Dialysis device

Lauri Lintott llintott at shaw.ca
Tue Nov 6 00:38:48 EST 2001


"D.K." wrote:
> 
> Lauri Lintott <llintott at shaw.ca> wrote:
> >BL wrote:
> >
> >> In additional to soft dialysis tubings and those membranes mounted on a
> >> frame, does anyone know of any other types of devices for dialysing a
> >> small sample? I need to dialyse a small sample which I don't want to put
> >> into a dialysis tubing or have to use a syringe to put into the membrane
> >> device (for fear of shearing the sample). A rigid tube with a wide enough
> >> opening (so that I can pipette in the sample) that has low adherence to
> >> proteins would be ideal. thanks for any suggestions.
> >
> >When working with small volumes of protein or DNA I use ambions 'microcon'.
> >(no personal affiliation).  They come in various mol wt cuttoffs.  Basically
> >to change salt concentrations or buffers you dilute your sample with the
> >buffer you are changing to, and then spin the sample.
> 
> >Everything below the
>  ^^^^^^^^^^
> >mol weight cut off spins through the membrane
> 
> This erroneous point of view appears to be widely held and is known to
> be a cause of many mistakes. I've seen a grad student who was
> seriously attempting to separate 50 kDa protein from 20 KDa contaminant
> this way (a completely hopeless and futile exercize). Truth is, cut-off
> value in both dialysis and ultrafiltration refers to a _retention_. As such,
> a cut off of 30 kDa does not mean that a 10 kDa molecule with go
> through with an efficiencty much higher that that of 30 kDa. All
> such a value says is that >95% of 30 kDa molecule will be
> retained (and less of 10 kDa molecule will be retained). It is prudent
> to keep in mind, for example, that even salts and things like glycerol
> and sucrose are concentrated to some extent by 10 kDa cut-off
> Centricons.
> 
> DK

Point taken DK, 
I was simplifying for the sake of message length.  Also, I believe that
microcons come with instructions about cutoffs, how to do buffer
exchanges, etc.  The original message did not say what was to be removed
by dialysis and I assumed that BL already understood the theory of
dialysis. I guess I should have said, "most of things larger then the
mol wt cut off will be retained, and almost of the salts will pass
through".

While there will be 'some' retention of salts, if you dilute say a 50 ul
sample into 500 ul, bring it down to 50 ul and then repeat, would not
this work as well as dialyzing against several buffer changes?  
Except for removal of amino acids from large proteins, or free dNTPs
from probes, I would not consider dialysis or centricons a good choice
for seperating macromolecules from each other.

Lauri




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