genecutl at mendel.berkeley.edu
Mon Apr 4 16:13:46 EST 1994
In article <wcg8744-010494111435 at pc15.hrfs.uiuc.edu>,
wcg8744 at uxa.cso.uiuc.edu (W. Chen) wrote:
> My friend is trying to purify a protein from plant leaf by the following
> procedure: The plant proteins were extracted by grinding the leaf in the
> phosphate buffer (pH 5.8). The crude proteins were then applied to a
> DEAE-Sephacyel column equilibrated with the phosphate buffer, and the
> column was washed with the phosphate buffer but no proteins were eluted
> out. It seemed that all of the proteins bound to the column. So the bound
> proteins were eluted with a 0-1 M NaCl gradient in the phosphate buffer. It
> was found that the total proteins eluted in a single sharp peak at an NaCl
> concentration of 0.4 M. The fractions of the peak were pooled and subjected
> to SDS-PAGE analysis and got the same pattern as the crude proteins prior
> DEAE-sephacyel chromatography. We don't undersdand why the proteins were
> not separated by the above procedure? If you have any suggestions or
> comments, please respond me at this account. Thanks.
The pH seems to be a bit low for phosphate buffer. Isn't the pI around
7.2? Phosphate doesn't have that great a buffering range anyway. Do
you know the pI of the protein? You might even want to try a negatively
charged column instead.
Also, if the proteins are coming off at .4 M NaCl, don't run the gradient
that steep, run it from 0 to .5M NaCl.
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