Centrifuge & Filter His-Tag Purification

 

This is the cheaper and easier purification method that doesn't require fancy machinery such as an FPLC. Purification occurs across multiple steps, but the final device to capture the protein is a filter that captures it before the storage buffer is added and the protein collected.

Equipment & Consumables:

  • Micropipette and Sterile tips

  • Ice in Ice Bucket

  • Wash Buffer

    • 50mM sodium phosphate (pH8.0)

    • 300mM NaCl

    • 10mM imidazole (autoclaved)

    • Using this buffer in conjunction with a second or third wash buffer with slightly higher imidazole concentration may help lead to cleaner purification.

  • Elution Buffer

    • 50mM sodium phosphate (pH8.0)

    • 300mM NaCl

    • 250mM imidazole (autoclaved)

  • Storage Buffer

    • 50 mM sodium phosphate buffer (pH 8.0)

    • 100 mM NaCl

    • 10 mM lysine (autoclave)

  • Nickel-Agarose NTA-Agarose Beads

  • Amplicon Centrifugal Filter Unit

  • 15 ml Falcon Tubes

  • Microcentrifuge tubes

  • Freezer

  • Protein in solution from lysis protocol.

  • Shaker in the cold (possibly inside old fridge)

Protocol:

  1. Invert the stock bottle of Nickel Agarose bead solution until the suspension is even

  2. Add 2 ml of resin to 10 ml Wash Buffer in a 15 ml falcon tube and invert to mix/equilibrate

  3. Centrifuge 2 mins at 3000g/4°C and then pour off supernatant (excess wash buffer)

  4. (Thaw if frozen from previous day, then) Add the 5-10 ml of cell lysate from the lysis protocol.

  5. Vortex the tube briefly to resuspend the resin.

  6. Leave to mix gently for ~20 minutes at 4°C to allow the his-tagged proteins to bind the resin.

    • No cold room, consider putting mixer in the fridge, or leave in fridge for longer with periodic manual shaking.

  7. Centrifuge 2 min at 3000g/4°C and discard the supernatant.

  8. Resuspend the pellet in 10 ml of Wash Buffer by vortexing and shaking.

  9. Repeat previous 2 steps twice more (3 x washes total). Discard final supernatant.

  10. Add 1 ml of Elution buffer and then vortex to resuspend.

  11. Leave to mix gently at 4°C for ~10 minutes, to allow his tags to elute (unbind) from the resin.

  12. Centrifuge 2 mins at 3000g/4°C.

  13. The supernatant now contains the target protein but also an excess of imidazole that will interfere with downstream processes.

  14. Label two microcentrifuge tubes "Eluted Protein plasmid-cell-incubation time-incubation temp-name-date" and set them up on ice.

  15. Transfer 20 μl of supernatant to one tube and freeze it with the other tubes for SDS-Analysis.

  16. Transfer the remaining supernatant to the other labeled tube and discard the left-over pellet.

  17. Assemble an even number (2 per sample is sufficient) of 'Amplicon centrifugal filter units' and add an equal amount of Eluted Protein solution to each (up to 500 μl max).

    • Assemble by putting inner tube inside of the outer tube.

  18. Spin for 15 minutes at max speed at 4°C.

  19. Discard flow-through. Add remaining solution (if any), then spin again for 15 mins at max speed at 4°C.

    • Note: Not all the liquid will go through the filter, but hopefully most of it.

  20. Add 400 μl of Storage Buffer to the top of the column (careful not to spill over the top if not enough has run through), spin for 15 mins at max speed at 4°C. Discard flow-through.

    • The protein should be trapped by the filter still, so the flow-through is waste.

  21. Repeat previous step twice more (3 rinses with storage buffer in total).

  22. Label a microcentrifuge tube "Purified Protein plasmid-cell-incubation time-incubation temp-name-date"

  23. Pipette the remaining solution on top of the filter (~100 μl) up and down vigorously several times to dislodge proteins from the filter, then pipette the entire solution from the top of the column into your labelled tube.

  24. This is the final purified protein solution. Quantify by A280, then proceed to the SDS PAGE protocol or freeze for later analysis.

Optional: Enzymatic Treatment for removal of Expression tags + C-peptide

This may be necessary depending on the design of your gene insert. For our insulin constructs, we use TEV Protease and Trypsin, but this is extremely experiment specific. Consult documentation on your enzyme if you plan on performing this step.

  1. Add 5 μl of protease stock solution (~1 mg/ml) per 100 μl of purified protein and then incubate at;

    • TEV: 30°C for 1 hour

    • Trypsin: 37°C for 4 hours

  2. Move the tube to the freezer, thaw when it is time for SDS PAGE analysis.

  3. These proteases will remain in the solution during the downstream analysis. They are difficult to purify once added at this late stage.

    • This could be solved by direct addition of the protease prior to the elution step. Note to self: test this later.

Acknowledgements: