I need to purify His6 tag containing protein and im using a Histrap that is been charged with Zn2+. Can i add DTT to my lysis buffer or the column will not tolerate it when i insert the lysate to it?
Thanks in advance
If you look up the Qiagen booklet about Ni-NTA, they mention the maximum concentration of DTT and mercaptoethanol tolerated in the column buffer (it is lower for DTT, which is a stronger reducer). Since Zn2+ has a lower redox potential than Ni2+, the same values should be safe for the Histrap loaded with Zn. Atr any rate, you could try a dummy run with the buffer and no protein and watch whether the aspect of the column changes.
It's easy to tell if you have reduced Ni2+ with DTT because it goes from blue to brown. Zn2+ has no optical absorbance, so you won't be able to tell if you have reduced it, assuming that is possible.
DTT usually interferes with any kind of IMAC resin. At least the traditional ones based on NTA and IDA ligands.
To overcome this, there is Ni-INDIGO resins that have a 20 mM DTT tollerance:
Just have a look at it. This way you should avoid any reduced metal ions during your His-tag purifications.
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/#features1
Hi,
It is recommended to use TCEP instead of DTT. Although the cost is higher, the range of resins that can be applied is wider.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column