Technique / Proteomics and protein biochemistry / Protein expression, protein extraction and protein purification


Troubleshooting affinity chromatography



Troubleshooting affinity chromatography

Affinity chromatography purification troubleshooting guide from Roche.

Problems:
1. Tagged protein appears in washes, does not bind to affinity resin.
2. Tagged protein remains bound to column, does not elute in Elution Buffer.
3. Extra proteins in eluant with tagged protein.
4. Tagged protein degraded.
5. Bubbles in affinity column.
6. Tagged protein elutes as a diffuse band.

Last update 28-Mar-2007, Rating of 3 votes.

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By man on 30-Aug-2008
Hi,
I am getting extra proteins in eluant with tagged protein even at 300mM Imidazole concentration during the Ni-NTA chromatograohy. When I run the Ni beads in SDS PAGE, I see my tagged protein is still bound to the beads and there are no other proteins other than my protein which remains associated with the Ni beads. How can I get rid of the other proteins. Should I go for even high concentration of imidazole, but then again I loose lot of my proteins.
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By susan on 06-Jul-2007
Hi pari,

It means your protein is not bound to the resin/Ni-NTA. You can try by increasing the volume of Ni-
NTA in column.

Besides for elution elution buffer should have comparatively high Imidazole concentration. You can try either of these.
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By pari on 28-Mar-2007
can u pls. advise me wht 2 do whn my protein which is His tagged comes completely in Flowthrough.My binding buffer has 50mM Tris pH:8,300mM Nacl,10mM Imidazole.
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