Dear All,
I want to know what is the basic advantage of using Ni NTA agarose over Ni NTA superflow for membrane protein purification? I know the capacity of the two are different, but is that the only reason?
I am trying to purify a membrane protein and after reading this wonderful discussion (https://www.researchgate.net/post/His-tagged_membrane_protein_doesnt_bind_to_Ni-NTA_column) here I am prepared to believe that Ni-NTA Spin kit (QIAGEN) is not a good option. Does anyone have any experience with this kit for membrane protein purification?
Thanks a lot in advance..
With regards,
Parijat
The binding efficiency of your protein of interest might be completely different. It only depends on the accessibility of the tag. There are plenty of examples where membrane proteins, once solubilized properly in detergent, bind pretty well to the Ni Beads. Your will have to test it. If you get good expression, find a suitable detergent that keeps your protein stable, you can be pretty happy already.
The difference of the agarose and the super flow is only the bead size. In case you are running a gravity flow column they should perform equally well. Me myself, i don't like the spin columns very much cause you easily dry them out.
In conclusion: just try and don't worry before you actually run into this problem
The binding capacity of Ni-NTA agarose and Ni-NTA superflow is the same. However, Ni-NTA superflow can be used at higher pressure and flow rate. Ni-NTA agarose could be used with gravity flow only but Ni-NTA superflow could be used with gravity flow and FPLC.
Usually the binding capacity of chelating sorbents is lower for solubilizating membrane proteins than water soluble proteins. It very strong depends from kind of detergents, concentration of detergents (CMC), salt concentration, b-mercaptoetanol or DTT supplements. Moreover purification of membrane protein is carried out at +4C by gravity flow for prevention of degradation. I advise to try all chelating sorbents during the same conditions and choose the best. There is no one right way. But on my experience Co2+-TALON is the best for GPCR purification.
Superflow runs at higher pressures, so your chromatography steps take less time. If you are doing gravity flow, you don't need that feature, so the advantage of agarose is just that the price is lower. Otherwise, they are almost identical (note, the capacity is not different). As suggested, membrane proteins have a lot of special needs during purification that have little to do with what kind of beads you are using.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression