I'm performing IMAC to purify my his-tagged protein (enzyme) from e. coli lysate. It seems I can get a good amount of my protein of interest but the problem is that I also get alot of coeluates, see attached file.
My protocol (everything at 4C):
Spin down 1L bacterial culture and sonicate the pellet in 50ml binding/wash buffer (20mM Tris, pH8, 20mM Imidazole, 500mM NaCl) --> spin down --> incubate lysate using 2ml nickel chelating resin from G Biosciences (786-281) overnight --> wash 4x 20ml --> elute in 9x 1ml increments in elution buffer (20mM Tris, pH8, 500mM Imidazole, 500mM NaCl)
After this I tried troubleshooting as indicated by G Biosciences: use less amount of resin, elute step-wise by increasing imidazole, reduce lysate-resin incubation time.
So I tried another experiment: 500ml bacterial culture sonicated in 25ml binding/wash buffer --> incubate lysate in 0.25ml resin for 1hr --> wash 4x 20ml --> elute in 7x 0.5ml increments in elution buffer with imidazole ranging from 100mM to 500mM (also incubated the flowthrough O/N with another 0.25ml resin and performed same experiment)
However, I still get background proteins. Any thoughts on this will be greatly appreciated.
Hi there,
You should add steps in your elution process between 20 and 100mM imidazole. What about the content of the flow through and wash steps. It sounds your final elution profile actually look like the input... Which is strange. The other thing you could do adding protease inhibitor cocktail to your lysis buffer in order to limit protein degradation during extraction.
Generally using IMAC we have a background concentration of imadazole in the all buffers and load/wash (did you add imidazole to the load sample?) etc this helps to reduce non specific binding (imidazole from 10-50mM have been used but is protein depend). Some researcher pre-incubate the column with imidazole (10-20 mM) prior to binding, this may also help. You could use a different metal ion say cobalt (or even zinc). All these metal ions generally have a lower affinity for Hist in order of highest Copper > Nickel=cobalt >zinc which may bind protein less stringently. You could even use a different buffer, (however we have found that Tris lowers the strength of binding to IMAC and may reduce background which is what you are using but it may well be worth a try)
Try washing with lower pH (pH 6 or less) and imidazole. I can't see any load and unbound samples on your gels, does all protein bind?
Thank you for your input. I did use protease inhibitor cocktail in my lysis buffer in the second experiment. I also used imidazole to lyse the sample in and to pre-incubate the resin with, both cases 20mM. Perhaps I should increase the concentration of imidazole in these steps as well to prevent background proteins binding to the resin.
Ian, can you recommend me a resin containing metal ions with stronger affinity for His? For now I will start by adjusting the imidazole concentration, if it doesn't help I will also start testing different buffers. About the pH, I'm not sure if I should do that since I'm trying to purify an enzyme and I need to retain its activity.
The company making the resin that I'm using also showed an example of protein purification on their website using the same resin. It seems there are background proteins in the elute as well (http://www.gbiosciences.com/ResearchProducts/NickelChelatingResin-desc.aspx) Perhaps its inherent to this resin?
Hi again,
In my lab we are using the Quiagen NiNTA resin which most of the times gives satisfatory results. We switch for NiNDA or Talon for difficult cases.
I see the company also sell zinc/cobalt chelated resins etc which might be worth a go and may reduce background. We buy in GEhealthcare 1-5ml Hi Trap columns (usually charged with Nickel, these are generally very cheap) but you can strip these and recharge with other metal ions. you can also purchase free resin from various places: Probond resin from invitrogen. It is probably worth just trying a couple of different metal ions.
But I agree use higher imidazole first. You want to get to a point where your protein does not bind efficiently then titrate back.
other additives: I would also include a detergent in your buffers such as Tween 20 at 0.1% or higher (up to 1%) to remove hydrophobic interactions, others have also used low concentrations of beta mecaptoethanol (10mM) in all buffer.
You could if need be take your eluted protein and put it back onto another IMAC resin (different metal ion) and bind/elute, or even a anion exchange resin and elute with a salt gradient. You could reverse this process, bind to anion exchange resin and elute in salt gradient. Pool relevant fractions and adjust salt to 0.5-1M and bind directly to IMAC.
bw
Thank you both. I will adjust my experiment and let you know how it goes.
Hi Amila,
Are you running the purification in batch mode? If so, I would try the column format. I've had good results with GraviTrap columns from GE Healthcare Life Sciences, as well as their Ni Sepharose 6 FF resin. I would also definitely try using more imidazole in the binding/washing buffer, 40-60 mM. Remember to include imidazole in the sample at the same concentration as the binding buffer. Good luck.
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