protein concentrating/desalting

Basavaraju Shankarappa 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.
Good luck
Raj Shankarappa
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.
> 
> Cheers
> -- 
> 
> bcrawfor at Sol.UVic.CA  -- member of the society for simple .signatures (SSS)
> 




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