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 CoHIS resin?
Many chromatographic procedures demand that the sample is pre-conditioned 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 10 mM β-mercaptoethanol can be used with the IMAC resin. Do not use strong reducing agents such as DTT or DTE as these tend to reduce the metal ion, which will lower the binding efficiency of the IMAC column.
5. How can I regenerate the metal chelate resin?
We recommend that you wash the resin with elution buffer and then re-equilibrate the resin binding buffer. Proceed to the preequilibration step if resin is to be re-used immediately. After regeneration, the resin can also be stored in a screw-capped bottle containing 0.1% sodium azide (made up in distilled water) at 2-8°C until further use.
6. Can I immobilize the metal chelate resin with a different metal ion?
It is possible to charge the resin with a different metal ion. Ensure that the resin is stripped of Co2+. This is achieved by successive washing with 1-2 column volumes of (i) 0.2 M EDTA, 0.5 M NaCl (ii) 0.2 M NaOH (iii) distilled water and finally (iv) 0.1 M metal salt. Then wash the column with at least 5 column volumes of distilled water to remove free metal ion.
7. What can I do if the resin has changed colour?
The pink colour is attributed to the Co2+ salt. Reductants (e.g. DTT) will cause the resin to turn discolour and chelating agents (e.g. EDTA) will cause the resin to turn white. Ensure that all solutions are compatible with the Amintra CoHIS resin.
8. How can I re-charge the resin with CoSO4?
Wash the resin with 3 column volumes of distilled water followed by 1 column volume of 0.1 M CoSO4 solution (made up in distilled water). Wash off any unbound CoSO4 with 5-10 column volumes of distilled water and equilibrate the resin with 1 x PBS buffer, pH 7.4.
9. 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.
10. 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.
11. 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.
12. 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.
13. Do I need to remove the His-tag from the recombinant protein after purification?
Normally, a protease cleavage site e.g. Factor Xa Protease is engineered between the His-tag and the target protein. The target protein can then be re-purified using Amintra CoHIS 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.
14. 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 Co2+, binding capacity may be reduced.