Ampholines and SDS-PAGE

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Feb 6 14:21:43 EST 2001


dfontani at facstaff.wisc.edu (Debora Fontanini) wrote:
:Hello,
:
:I am puzzled by some results I have been getting and I was hoping some of=20
:you could shed some light on this.
:I am running on SDS-PAGE (12.5%, silver stained) a protein sample that was=
:=20
:eluted from a PBE 94 column (pH range 4-6).  The ampholines that are=20
:present in the Polybuffer used for the elution, should not bind to the=20
:proteins and I though I could quickly get rid of them using a 500-=B5L=20
:concentrator which at the same time allows to exchange the buffer.  The=20
:sample so concentrated, was separated on PAGE and when stained for=20
:proteins, all I could see was a bunch (4-5) of bands with very similar=20
:migration rates, very closely spaced.  My protein should be relatively pure=
:=20
:at this stage and although I do not know if it has isoforms, I am wondering=
:=20
:if the ampholines could have had something to do with the appearance of=20
:multiple bands.
:A collegue of mine is also using the same procedure (PBE 94=20
:chromatofocusing) in the course of a purification; she also sees a similar=
:=20
:phenomenon on her silver stained gels.
:

What you see is most likely indeed ampholites. Many of them are
quite high in MW. Microconcentrators that you used _do  not_ 
effectively get rid of LMW substances. Just like with dialysis tubing,
the cut-off specified tells what MW will _not_ pass, and that does not
mean that anything above will (much less "quantitatively" will pass 
through). 

In fact, even Centricon-10 (10 kDa) even concentrates salt and glycerol 
to some extent. 

Do a mock concentration run from your elution buffer and see if
such sample gives you the same bands on SDS. My bet is that it will.

        - Dima






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