Hello everyone. Long story short, I am struggling to purify a soluble protein which has a 6X His tag. I ruled out the issues with the expression vector, as well as faulty induction (i.e small scale expression went fine and showed up on the SDS PAGE).
I elute the protein with Imidazole 250mM using 3 buffers with varying pH and the gel shows that it gets stuck on the Ni resin with no protein at all (not even faint bands) in the elution fractions. The protein is not too stable so I don’t want to experiment with pH a lot. Should I increase the concentration of imidazole? What is the reasonable concentration of it for elution which won’t complicate the further purification and quantification (BCA assay will be used).
Thank you very much!
No protein in the flow-through? What adsorption buffer are you using? What pH values have you tested? Is the column pressure increasing?
I use 500 mM Imidazole (pH 7.5) with 50 mM NaCl to elute my protein (bacterial cytochrome P450). So I think you can try increasing the concentration of imidazole. But again you have to get rid of the excess salt and imidazoles by using a desalting column. Also consider isoelectric point of your target protein, since you mentioned it gets to the Ni resin.
With 6xHis, your typical protein will elute at 150mM Imidazole rather quantitively, unless it has a high amount of histidines itself which strengthen the binding. With 250mM Imidazole, I got to elute pretty much any 6xHis Protein to completion this far. Some go to 500mM but seems unreasonable to me unless you got some stuff like 12xHis (9xHis for the most part retains at 75mM Imidazole, though this always depends on the length of these washing steps as your POI lets loose bit by bit).
When eluting with imidazole, don't vary the pH too much, there is no reason to. Depending on your pH, you might be too near to your protein isoelectric point, leading to precipitation. As IMAC resin doesn't work below pH7 (pH5 is typically used for pH-elution), make sure you are 7.5-10. If the pH is too high, your resin will reversibly turn to mud-green Ni(OH)2 while losing binding capacity.
In case you're not sure if the protein still clings to the resin, simply boil up a portion of the used resin in SDS-PAGE loading buffer.
Did you boil the beads in the SDS-PAGE loading buffer and then run them on SDS-PAGE to see whether your protein is bound to the resin? Also, what kind of three buffers did you use?
I have used 50 mM - 250 mM Imidazole in native buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 10% Glycerol and 2Mm PMSF protease inhibitor] for the elution of the proteins, which worked perfectly fine.
Thank you for your reply. Yes, I did, and the protein band shows up on resin beads only on SDS-PAGE. Could you please tell me why you used glycerol for elution?
Well your protein is precipitating due to the adsorption per se or to its unstable nature. You need to improve this.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials
- Polymers