The protocol of Thermo Scientific NiNTA resin (cat. 88221) for purification of proteins containing a hexahistidine tag use an elution buffer containing 250 mM imidazole. For fun after this elution I put 3 mL (for a 1.5 mL bed column) of 1 M imidazole, and on SDS-PAGE I see a lot of protein eluting, which means low recovery yield using only 250 mM.
Is there a problem using higher amounts of imidazole? I've seen is common to use 500 mM, but what about 1 M or more?
Dear Luis
Which volume of 250mM buffer did you used for elution?
Generally i'm using a 300mN imidazole for elution and in 3-6 CV (that in your case mean 4,5-7ml) generally I acheive complete elution of my proteins.
In principle you can certanilly increase the imidazole concentration in the Elution buffer but you have to consider:
1) costs
2) It may affect some step as for example quatification. High imidazole concentration for example provide backgorund at bradford or at 280nm if you do not buy imidazole with high purity levels.
3) You have to remove it after the purification or this concentration is compatible with your next step?
good luck
Manuele
300 mM imidazole for elution of target protein and 500mM for washing column (elution of unwanted material)
- Badges
- Science method