Elution of GST fusion protein

Tupy.Jon tupy at rapids.tularik.com
Sun Feb 6 15:46:08 EST 1994


Hi netland,

I have produced a GST fusion protein via T7 promotor in e. coli (BL21) that is
about 70 Kd (including GST).  After an initial fractionation on SP Sepharose, I have
attempted a secondary purification on Glutathione Sepharose 4B (Pharmacia). 
While I have obtained some pure fusion protein in eluting this column, the vast
majority (>90%) of this protein remains bound to the column.  My elution buffer
contains 20 mM reduced glutathione,100 mM KCl, and 25 mM HEPES (pH 7.9).  I first
applied this buffer at 4 degrees C, which didn't work.  Then I attempted an
elution at room temp, which also didn't yield additonal protein.  Finally, in an
effort to batch-elute the fusion protein from the resin, I added two column
volumes of elution buffer to the resin, capped the column at both ends, and
incubated the whole thing on a rocker at RT for an hour.  *Still* no elution!

Anyone have any tricks?  Thanks!

Jon



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