I'm trying to purify the protein by Ni-NTA affinity chromatography. It seems that my protein (size about 54 kDa) is co-purified with chaperone protein (probably GroEL - as the band is around 57kDa). I tried the method where 4M glycerol and ATP is added to the wash buffer 1 and then I incubated this with the resin for 2 hrs in 4C, but it did not help. Has anyone met such a problem before? I will be grateful for any advices.
If chaperone proteins are not an expected binding partner to your protein/peptide fragment then you need to use imidazole in your lysis and wash buffer ranging from 1 to 10 mM depending on how stringent you want but 1 mM worked for me. Can go higher than 10 mM if the problem does not resolve but don't go too high since imidazole is also used to get the purified proteins off the nickel beads as an elution buffer!
Is it possible to clone your gene of interest onto a new plasmid (preferably with a GST/avidin/alternative tag)? If all else fails, this would probably remove the problem with elution.
protected by Chris♦ Dec 19 '14 at 12:07
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