Yeah you can try all of these you mentioned, so long as they don't affect your enzyme activity that much.

You can also adjust the ionic strength of your wash buffer, by increasing/decreasing salt concentration.

What do you use for elution? Acid or imidazole? You can add varying amount of this to your wash buffer, in order to minimize non-specific binding.

Best.

Hello,

in principle you can try any of the listed chemicals, but be careful to denaturing the protein, especially in case of urea or guanidin. However, it sometimes helps to elute the proteins by a shallow gradient of imidazol, as in the case of IEC, provided that the proteins have different affinity to the resin.

Marek

did you firstly wash with 50mM imidazole before elution ?

Do you work with membrane proteins ? In E. coli, AcrB binds naturally to HisTrap column.

So you have to elute your protein with a gradient.

Hi, thanks for all the answers! (A/B) buffer is: 100 mM Tris, 0.5 mM NaCL and 25/500 mM Imidazol, pH=8. I elute over 10 CV, and gel of protein expression look like this. HisTrap is last 6 lanes, at the 3rd marker band. Marker is: LMW (97, 66, 45, 30...). L1: Marker, L2: cell growth, L3: after IPTG, L4: sonification supernatant, L5: Resuspended pellet, L6: flow trhough, L7-L12: HisTrap. So, I have a lot of the protein going into the pellet, but until now, we still just work with the supernatant

Selecting last 3 fractions, i do AEC (20 mM tris, pH=8, 25/500 mM NaCl). That traps me on both sides after AEC. So I was hoping to include an additional wash step in the IMAC.

I have heard about washing with triton x-100, but not many good outcomes. Has any had good success?

Are these two proteins of such big concerns? If so, just use some other column like anex or maybe size exclusion, if they are of different size.

I don't see any reason, why should Triton improve your purification on HisTrap or why should it elute anything from such column.

I was thinking that the unwanted proteins could bind to my protein by non-specific or hydrophobic binding. Or it could be the proteins was some stress responsive proteins. And Triton by being a detergent, could maybe remove it. I simple does not understand why other proteins can have such an affinity for the HisTrap column.

You could also try increasing the ionic strength or [imidazole] in your loading buffer, and/or including detergent, to prevent the contaminants from binding in the first place. Do you know what your contaminants are? if they are chaperone proteins, that coudl be telling you something about the "health" of your protein. Depending on your expression level, using a smaller column may also help reduce non-specific binding. The QIAGEN handbook also has a pretty comprehensive list of things to try.

The proteins may simply have several amino acids capable of binding to Ni2+ on its surface. Thus it binds to the column, not to your protein.

The idea of chaperones has also come in to my mind. I looked at DnaJ(HSP40) and DnaK(HSP70). The amersham handbook recommends burst of 10 seconds in sonification. At the moment I have used 3x1 min burst, with 30 seconds pause. Have others experienced that sonifacation can have a huge impact, and lead to this elution pattern? I would like to identify the other proteins, but time is money. :-)

Hi Troel,

Have you ever try ion exchange chromatography by pH gradient elution?

regards

K

If you are considering guanidinium or urea in your wash, then does this mean you don't need your protein to be active? If so, then you should do the prep under denaturing conditions, which are better for separating tightly bound proteins.

However, if you are trying to purify active protein, you can try washing off bound chaperone proteins with ATP. Also, try expressing your protein at room temperature instead of 37C.

Use 50 mM tris-HCl, 7.5 pH instead of 8.0 and 500 mM NaCl including 50-70 mM imidazole in binding buffer. You can use 1M NaCl to prevent other weak interactions with E. coli proteins. If it doesnt work, use 1,2 ,3, 4 M Urea wash after binding of your protein to the column and monitor in each step to find best conditions. Sometimes oversonication is problem, you can open cells by using lysozyme, it may help. As suggested by people, you can use ion-exchange, but if the complex is tight they will eluted in the same gradient. Gel filtration will not help becuase proteins are very close (high mol weight), or similarly if they are in complex, they will come as single fraction.

If nothing works, Use cell free expression system to get rid of all problems.

It is also important to know about the concentration of imidazole in which your gets eluted. Addition of imidazole (preferably 10-25mM) to the wash buffer may help you to get away with the intruders in your purified fraction. You can try increased volume of wash buffer to reach at a perfect point of elution of your protein. All the best.

Hi Troels,

If contaminants bind to the His-Trap media : Like some said, adding imidazole in loading buffer (up to 40-50 mM) improves greatly the non-specific binding on Nickel, and is far more efficient than trying to separate your protein from the contaminants after binding. Another point : do you have a specific protease site between his-Tag and your protein ? If yes, make the His-Trap, cleave with the protease and change the buffer towards 20 mM Imidazole, and do a second His-Trap. Your protein will be in the column flowthrough and the contaminants and the Tag will remain bound on the column.

If contaminants bind to your protein and not to the media : trying a high salt wash (1 M NaCl) with no or low imidazole can remove ionically interacting proteins, and a wash with detergents and low ionic strength can remove hydrophobically interacting proteins...

Good luck !

Thanks for all the answers! I think I will close it here.

I will try some different things now, and write how it goes.

Try to change the sonification time to shorter burst.

Add a 2% triton wash step.

Change the binding/elute buffer to: 50 mM tris-HCl, 7.5 pH, 0,5 M NaCl and 50/500 mM imidazole.

Thanks!