Hi I am trying to purify a complex, GST-tagged protein. This protein is prone to aggregate and unstable. My column was packed using Sepharose 4B.
My question is :
I found almost all protocols recommend 10-20mM reduced Glutathione, 50mM Tris pH8.0 as Elution buffer.
Why is there no NaCl in Elution buffer?
Cause I am considering to modify Elution buffer below to make my protein stable and active but not sure whether I am on the right direction.
50mM Tris pH 8
10mM reduced Glutathione
20mM NaCl
10% glycerol
5mM b-me (No DTT because overnight sample loading)
The glutathione-Sepharose 4B instruction booklet has this to say:
Pack the gel in any suitable Pharmacia chromatography column.
•Wash the column with 5-10 bed volumes of PBS (140 mM NaCl,2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) to remove the
preservative.
•Equilibrate the gel bed with 3 bed volumes PBS.
•If needed, clarify the sample by centrifugation and filter thesupernatant through a 0.45 μm filter before applying to thecolumn. The sample may be diluted in PBS + 1% TritonX-100,if too concentrated.
•Apply the sample to the column and discard the eluent.
•Wash the column with 5-10 bed volumes of PBS.
•Elute the bound material with 5 bed volumes of elution buffer
(10 mM Glutathione in 50 mM Tris-HCl pH 8.0) and collect the
fractions.
source: http://www.ritsumei.ac.jp/~h-matsu/GSTmanual.pdf
I suppose that this is why people usually report using that elution buffer. But notice that the equilibration buffer (PBS) contains 140 mM NaCl. So there doesn't seem to be any reason to avoid using a bit of NaCl in your buffer if you think it will help keep your protein from aggregating.
Hi Chen, you probably meant to say that your column was packed using Glutathione Sepharose 4B, not plain Sepharose 4B.
Anyway, if your protein is prone to aggregation, it could be sitting on the top of your column.
There's no way 20 mM NaCl would hurt. I'd be much more worried about your 10% glycerol. That might give you some very uncomfortable back pressure and slow down your binding kinetics. Try to decrease that and running it real slow, maybe, good luck.
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