Protein purification?

gc genecutl at
Mon Apr 4 16:13:46 EST 1994

In article <wcg8744-010494111435 at>,
wcg8744 at (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
> to
> 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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