protein concentrating/desalting

Dennis J. Templeton djt2 at po.CWRU.Edu
Wed Feb 17 10:48:55 EST 1993


In a previous article, bcrawfor at sol.UVic.CA (Bryan Crawford) says:

>I have a little technical problem which I would like to solicit
>net.advice on.
>
>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

Amicon (and some other companies) make spin columns with MWCO filters
(that's molecular weight cut off, like ultrafiltration)  You put your
sample in the reservoir and spin the tube, and molecules smaller than the
cut off will pass through.  Larger molecules are retained above the filter.

The medium sized ones are called centricons, with a reservoir of 2-4 ml I
think, and there are ones that fit in microfuge tubes called... minicons I
think.  the minicons have a hold up volume of less than 50%, and are
supposed to be very low protein binding.  I haven't tried it with nanogram
quantities though.

Cut offs run from 3kd to 30 kd, I think

have fun,

dennis



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