1. What is the shelf-life of Metal Chelate resin?
The resin is guaranteed for 2 years after the date of manufacture provided they are stored at 2-8˚C.
2. Do I need to filter the buffers prepared in my laboratory?
It is good laboratory practice to filter all buffers.
3. How should I prepare my sample for the Amintra NiNTA resin?
Many chromatographic procedures demand that the sample is preconditioned prior to loading. We recommend that all samples are filtered. High viscosity is mostly attributed to contaminating DNA or RNA. The intrinsic viscosity of a lysate can be reduced by either drawing it through a syringe needle several times or by adding appropriate amounts of DNase and/or RNase (5-10 µg/ml) to the lysis buffer and incubating the mix on ice for 15 mins.
4. Should I add b-mercaptoethanol to the lysis buffer?
Reducing agents can reduce the resin matrix and adversely affect binding of the His-tagged protein to the resin. Its inclusion depends upon whether the His tagged protein elutes with contaminants as β -mercaptoethanol can reduce all disulphide bonds formed between the contaminating proteins and the target protein. We recommend 0.5mM TCEP. Concentration less than 20 mM β-mercaptoethanol can be used with the IMAC resin. Please see compatibility table for more detail.
5. Can I immobilize the metal chelate resin with a different 7. What can I do if the resin has changed colour?
The blue colour is attributed to the Ni2+ salt. Reductants (e.g. DTT) will cause the resin to turn brown and chelating agents (e.g. EDTA) will cause the resin to turn white. Ensure that all solutions are compatible with the Amintra NiNTA resin.
6. How can I ensure that levels of contaminants in the final eluate remain low?
Ensure that the binding buffer contains minimum 10 mM imidazole and the wash buffer contains minimum 20-30mM imidazole.
7. Should I be concerned if the resin partially dried out during the chromatographic steps?
The resin is robust. Partially dried resin rehydrates rapidly. There are no adverse effects upon the performance of the resin.
8. Should I remove imidazole after the final elution step?
Imidazole is best removed after elution if the protein is going to be stored. Otherwise, the protein may precipitate out of solution at -20 or -80ºC. Alternatively you can use a Stabil-PAC kit to enhance protein stability in imidazole solutions.
9. Can I load purified protein immediately on to an SDS-gel?
Proteins purified under native conditions can be loaded on to an SDS-polyacrylamide gel. Those proteins purified under denaturing conditions in 6-8 M urea can also be loaded directly on to a denaturing SDS-polyacrylamide gel. Proteins purified in the presence of 4-6 M guanidine HCl should be buffer exchanged in buffers lacking the denaturant prior to a denaturing SDS-PAGE.
10. Do I need to remove the His-tag from the recombinant protein after purification?
Normally, a protease cleavage site is engineered between the His-tag and the target protein. The target protein can then be re-purified using Amintra NiNTA resin in order to purify undigested His-tagged protein. For most applications, it is not necessary to remove the His-tag. However, it is often desirable to remove the His-tag if X-ray crystallography or NMR is to be used to determine the structure of the target protein. When protein precipitation is observed during cleavage Expedeon’s Stabil-PAC (# STP) can be used to maintain protein solubility.
11. Can I re-use the resin?
The resin can be re-used. Re-use does depend on the properties of your target protein. You may observe that flow rates slow down in successive bind-wash-elute cycles as more samples are progressively loaded on to the columns. In addition, if the resin is not re-charged with Ni2+, binding capacity may be reduced.