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Resin plug loading

1. Load the pre-packed resin Midi plug containing immobilized IMAC resin into the Proteus spin column barrel using the Midi insertion tool as shown on page 15.

Pre-equilibration (Total spin time = 3 mins)

2. Equilibrate the IMAC spin column with 10 ml binding buffer, pH 7.4 (10 mM imidazole) by centrifuging the spin column at 500 g for 3 min.

Clarification of sample

3. Filter 22-25 ml sample first through a 1.2 µm pore size syringe filter and then immediately afterwards through a 0.2 µm pore size syringe filter to remove any cellular debris, precipitating protein complexes just prior to sample loading.

Sample loading (Total spin time = 30 mins)

4. Pipette up to 20 ml filtered cleared lysate into the spin column and centrifuge the spin column at 100-150 g for 30 min. It may be necessary to increase the spin speed or spin time if any sample remains above the plug. N.B. In some circumstances, you may wish to re-apply the sample wash back through the spin column before the wash step in order to increase the residence time between the target protein and the resin plug for efficient binding.

Washing (Total spin time = 9 mins)

5. Wash the spin columns up to three times with 10 ml wash buffer pH 7.4 (30 mM imidazole) to remove non-tagged proteins with no affinity for the immobilized metal ion by centrifuging the spin columns for 3 min at 500 g. The washes should be collected for analysis. As imidazole absorbs UV radiation at 280 nm, we recommend that the wash buffer is used as the reference solution for auto-zeroing the UV-Vis spectrophotometer if imidazole is used to elute the target protein from the spin columns.

Elution (Total spin times = 6 mins)

6. Elute the bound His-tagged protein with 10 ml elution buffer pH 7.4 (300 mM imidazole) directly into a fresh centrifuge tube by centrifuging the spin columns for 3 min at 500 g. The eluate should be collected for further analysis. Repeat the above elution procedure faithfully to ensure complete recovery of all recombinant proteins.

N.B. Check the protein content of each eluted fraction before pooling them. Otherwise, you risk diluting a concentrated, purified sample.

Desalting and concentrating

7. Imidazole and any residual metal ions should be removed by diafiltration using ultrafiltration concentrators or rapid dialysis against an appropriate buffer for your downstream application. Otherwise, the imidazole may strip the metal ion from a metalloprotein of interest or the target protein may irreversibly precipitate out of solution when stored at –20 °C or –80 °C.

Regeneration of the IMAC midi plug

8. Wash the Midi plugs with 10 ml elution buffer by centrifuging the spin columns at 500 g for 3 min. Then wash the plugs with 10 ml binding buffer by centrifuging the spin columns at 500 g for 3min. Proceed to the pre-equilibration step of another bindwash-elute cycle if the plugs are to be re-used immediately. After regeneration, plugs can also be stored, without their end caps, in 0.1 % sodium azide (made up in distilled water) at 2-8 °C until further use.

Easy to read midi purification protocol:

 

Easy to read midi regeneration protocol: