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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 Fair of 2 votes.

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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.
Rating: Good

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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.
Rating: n/a

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