The other fusion proteins are eluted well. But there is something wrong with only one protein. It is expressed well in 500 ml LB with 0.2mM IPTG, overnight incubation at 20C and 150rpm. The problem is elution. The elution buffer contains 500mM imidazole and pH is 6.0. I am looking for a solution and some recommendations.
Hi
The reasons may be several, as well as the solutions for your problem. You can try increasing the imidazol concentration up to 1M or change the metal ion for reducing the interaction affinity. Alternatively to the elution by competition, you can try to elute your protein at pH 4.5 (of course, if your protein is stable under these conditions). At this pH value, histidine residues change their electronic configuration making them unavailable to complete the secondary valencies of the immobilized metal ions. So, no imidazol is needed in the elution buffer.
So What if i want to include EDTA in my elution buffer? What molar concentration of EDTA would be enough for elution ? Also Can i remove EDTA through PD10 column?
Yes, you should remove it efficiently through pores with cut-off of 10 kDa.
50-100 mM EDTA is used to remove all the metal ions from the resin in stripping protocols, so these conditions can be used to elute your protein. EDTA can then be removed from your sample protein by PD10 or dialysis. Just remeber to recharge your column with fresh metal to re-use it.
Thank you very much for the answers. I have a last question in my mind. Is there any limit for PD10 Column to desalt EDTA? I mean tolerance of PD10 for EDTA?
if i add EDTA to my elution buffer then my elution buffer will be a mixture of 500mM imidazole and 50mM EDTA? Is it fine to use both in a single buffer ? and Also could it be desalted on PD10 column? Please give your input about this question. Thanks again and waiting to hear from all of you.
What do you mean by PD10 column? Is that chromatographic desalting column? If the cut-off is 10 kDa, you're far below the limit with EDTA.
As said elsewhere, you do not need to use both imidazole and EDTA.
Are you sure the protein stayes on the column? Did you measure activity in flowthrough? Did you try to elute from the column with urea and check on Western/SDS-PAGE?
Tomas Hluska thank you so much for your useful suggestions. Well i tried to elute my protein wih three different buffer system.
a) 500mM imidazole buffer
b)500 mM imidazole + 50 mM EDTA
c) 50mM EDTA only.
500 mM imidazole gave very weak elution.
500mM imidazole + 50mM EDTA worked better. eluted well compared to 500mM imidazole alone.
50mM EDTA didn't give any expression at all.
After elution i desalted the eluted product using GE Healthcare PD10 column with G-25 medium.
Now i need to confirm whether my eluted product will be having EDTA or not. Any simple idea by which i could try it.
Thanks again.
Before you proceed with analysis of the elution, make this:
1) measure activity (or whatever you use to determine your specific enzyme content) in the sample you load on the nickel column
2) measure activity (see above) in what didn't bind to the column
3) count TOTAL amount of your enzyme in the load (No 1) and flowthrough (No 2). Does it differ by at least 10%?
Simple yes/no. Otherwise it doesn't make sense to proceed.
It seems a good idea to determine the efficiency of the IMAC step by estimating the percentage of (your) protein you can recover after elution in comparison to the initial amount loaded onto the column.
About the PD10, EDTA, imidazole (and also an important part of the Ni striped out from the column) should be easily removed from your protein. Just take care of use the recommended volumes to elute your protein and for washing off the salts between loadings cycles. If you need your protein being completely EDTA and Imidazole free, discard the last 250-500 uL from the PD10 elution, wish are more prone to be contaminated with the beginning of the low-MW components.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression