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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.

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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!

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  • $\begingroup$ My lysis buffer and first wash buffer contained 10 mM Imidazole, then I made second wash with 10% buffer B in A, and eluted protein with buffer B contained 250 mM imidazole. The band of chaperone protein appear in 'second wash' fraction and 'elution' fraction but mostly in 'elution' fraction. $\endgroup$ – Ala Jun 10 '14 at 23:29
  • $\begingroup$ The go higher, try 25 or even 50 mM in your wash buffer (and maybe lysis buffer). I know someone had this very exact problem and increasing imidazole concentration solved it! I can't remember the exact conc they used but it was between 20-50 mM (that I remember for sure!). However it might effect your purification yield, but thats the compromise you might have to make. If you have high amounts of purified protein, then it shouldn't matter! $\endgroup$ – Bez Jun 10 '14 at 23:35
  • $\begingroup$ Also try increasing your wash numbers since two is far too low! Try 5 perhaps. I do 3-5 washes. $\endgroup$ – Bez Jun 10 '14 at 23:44
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    $\begingroup$ Also in what system are you over-expressing your protein since what you are seeing at 47 could be the result of post-translational modifications (embl.de/pepcore/pepcore_services/cloning/…), which can increase the MW of your protein from 54 to 57 kDa! i.e. what I'm saying is how do you know you are co-puryfying a chaperone? $\endgroup$ – Bez Jun 11 '14 at 0:11
  • $\begingroup$ I did western blot, so I know which band is my protein - it is below the bigger band. It is T7 expression system which is started by adding IPTG to medium in final concentration of 0.1 mM. $\endgroup$ – Ala Jun 16 '14 at 20:51
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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.

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