I am trying to search for a better way to purify my histidine tagged protein by looking for a replacement for EDTA, since it trips the metal ions in de IMAC columns. I first used the Nikkel column but want to use the Cobalt column since this is more specific for histidine. But I want to take it further by looking for a replacement for EDTA. And when replacing it, do I need to use an other buffer? I am using the TRIS-HCL buffer with pH 7.5.
You are using a high edta concentration. 2 mM should be enough.
You can also use imidazol instead of EDTA for elution. You can apply a gradient elution with imidazol, for example from 0 to 100 mM imidazol. An advantage of imidazol is that it leaves the bound metal on the column and only elutes the protein while EDTA also detaches the complex forming metal ion.
Peter
Omar, I have been using an 5 mM EDTA concentration, is there a way to make a coctail of it in combination with other protease inhibitors? Or is 2 mM just enough to use?
Peter, I have been using imidazole in my experiments. I started with 30 mM start buffer and went to 70 mM. Then jumped to 125 mM and then to 250 mM. What can I change in these steps?
As mentioned above you can try a gradient first and trace the elution. From the profile information you then can select suitable concentration steps for further experiments.
And is there a way to adjust my lysis buffer for when I work with histidine tagged RAD51 protein? It contains the 5 mM EDTA, but I want to either lower the concentration EDTA to 2 mM or replace it with DTT and leupeptin and/or benzamidine protease inhibitor. I want to try and find a way to use less metal chelator when working with IMAC and still get quality results.
2 mM is enough to inhibit metalloproteases. If you have other kind of proteases then switch to an expensive cocktail.
For an IMAC run your sample generally should not contain any EDTA as it detaches the immobilzed nickel or cobalt from the column and just preventing or diminishing binding of your his-protein. There are several methods to get rid of the EDTA such as dialysis or ion exchange chromatography (column or batch). It is also possible to counterbalance the effects of EDTA by addition of equivalent amounts of Mg2+.
Peter
I will try to keep the concentration at 2 mM, and before the purification I will counter it with 2 mM Mg2+, all this while using a cobalt column this time instead of a nickel column.
Thank you for the info Omar and Peter ;)
I have a His-tagged protein that I purify using Tris buffer at pH 7.4. I do not use EDTA since my is a metalloenzyme. Protease cocktail inhibitor or PMSF works just fine.
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