can i autoclave buffers with imidazole for His-tag purification? - (May/19/2005 )
I purify a pentamer protein on the Ni-NTA superflow resin (qiagen). In Western (after SDS PAGE) I have a beautiful band, but after Ni column (elution with 50 mM Tris pH7.9 + 0.5 M NaCl, +imidazole from 5mM to 2M), - nothing. Can therefore imidasole-containing elution buffer be autoclaved? (to kill eventual proteases?)
What other possibilities can i try?
i think you have to drop the Ph of elution buffer to 5.9 or below that. and also u can autoclave the tris, NaCl and after u can add imidazole without autoclaving it.
better filtering them on 0.22µm filter.