bsh at MED.PITT.EDU
Wed Feb 17 10:25:55 EST 1993
I have used one small trick that might be of some help in reducing
your loss in dialysis. Instead of loading a dialysis tubing with the
sample, cut out a piece of the dialysis tubing, wash it as usual, then
slit it so that you now have a dialysis sheet. You can now float this
dialysis membrane sheet on a petridish filled with the buffer you want
it dialysed against.
Now place about 50-100 ul of your sample as a drop on this floating
piece and let it sit for about an hour or less. You can carefully draw up
the sample and it should contain significantly reduced salts.
I remember seeing this technique from some literature. I cant
remember where. Since you are not placing all your sample in the bag
where I think you lose most of your sample, I think this method is much better.
You can make several floating castles when you have more volume.
Hope this makes sense and you may want to try.
bsh at med.pitt.edu
> I'm using an affinity column to purify 125-I labelled cell surface
> material from sea urchin cells. Since the buffer the eluent is collected
> in is very high in salt, I can't concentrate my samples and run them on
> SDS-PAGE right off...I have to desalt them and concentrate them. Because
> I am collecting very small quantities of labelled protein (<10ng), I can't
> TCA precipitate and resuspend in PAGE sample buffer :-(
> I have also tried running the samples over a desalting column, and then
> concentrating the eluant, but find I am loosing a too much of my sample
> in the manipulations.
> Simple dialysis, followed by concentration by vaccuum centrifugation works
> okay, but again, my yeild is poor (loosing about 70% of my counts, sticking
> on the dialysis membrane).
> Can anyone suggest any clever tricks for concentrating and desalting extreemly
> dilute protein solutions? (I though of using a butenol concentration which
> I know works for nucleic acids, but I don't know if this will take the salts
> out, and I'm not sure it will leave my protein behind).
> Any and all suggestions greatly appreciated.
> bcrawfor at Sol.UVic.CA -- member of the society for simple .signatures (SSS)
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