pI=9.5 protein will not bind to cation exchanger at pH 6.5

Roland J Saldanha rsaldanh at magnus.acs.ohio-state.edu
Tue May 3 19:35:31 EST 1994


In article <2q6job$21t at news.iastate.edu>,
Bipin K Dalmia <bipin at iastate.edu> wrote:
>i'm trying to purify a protein expressed in e. coli. there is tons of it
>in the soluble extract. the pI is about 9.5. so naturally i tried using
>a cation exchanger at pH 6.5 but my protein flows right thru it. i've
>checked the ionic strengths etc. and they look good. the resin was
>properly equilibrated too. i'm using bio-rad's biorex-70 resin in the
>sodium form and eluting with a gradient of NaCl.
>
>any clues?
>
>bip
>--
>bipin k. dalmia               the other night i was lying on my bed, looking
>bipin at iastate.edu             up at the beautiful stars, and i said to myself,

>n2.bkd at isumvs.iastate.edu     'where the F*CK is my ROOF !!'
>--


Frequently this is due to nucleic acids contaminating a prep.  Basic proteins 
stay tightly bound and their charge is thus masked and the protein flows 
through.  If you have not already treated your extract to remove nucleic acids 
you might consider:
PEI precipitation in high salt (0.5M) to remove nucleic acids followed by 
ammonium sulphate precipitation of the protein to get rid of the PEI.
or nuclease treatment or gel filtration in high salt (if your protein is small 
enough to seprerate from bulk nucleic acids).
PEI is by far the most widely used.



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