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Nickle Agarose Regeneration Protocol

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to regenerate spent Nickle Resin with new unused Nickle.

Reagents and Materials

Things to Note before starting

Calculating CV

Measure the height of the blue Nickle Resin that settles after the column has been washed. The tubes we have in the lab are 4.9cm2 in surface area, so multiply the height of the Nickle Resin in cm by 4.9 to get the CV in mL.

Waste

None of the flow through from the column needs to be saved to be analyzed. However, it should be saved in various collection tubes and disposed off by contacting EH&S.

Water

Ideally, we should be using NanoPure Water for all the steps, however that is extremely expensive. Go to the Strong Lab (3rd Floor Weintrub Building) and use their MilliPore DI Water dispenser to collect 4L of water in our water jugs.

Estimated Timing

This also takes a long time, sometimes up to 10 hours. This is definitely something you should do over the course of multiple days.

Procedure

  1. Vortex the Used Nickle Resin to completely resuspend Nickle.
  2. Add 50mL of the Used Nickle Slurry to as many columns you can.
  3. After the resin settles, calculate the CV of each column.
  4. Wash the columns with 5CVs of H20.
  5. Add 2CVs of RM034 - List of Commonly Used Lab Buffers#50mM EDTA|50mM EDTA. This should cause the blue Nickle to leave the resin.
  6. Wash with 5CVs of H20.
  7. Cap the column and add 2CVs of RM034 - List of Commonly Used Lab Buffers#0.5M NaOH|0.5M NaOH. Let the NaOH incubate for 25 minutes.
  8. Add 20CVs of H20 to neutralize the NaOH.
  9. Add 3CVs of RM034 - List of Commonly Used Lab Buffers#100mM NiSO4|100mM NiSO4 to regenerate the Nickle.
  10. Wash with 10CVs of H20.
  11. Cap the Column and resuspend the regenerated Nickle with RM034 - List of Commonly Used Lab Buffers#20% ETOH|20% ETOH.
  12. Transfer the regenerated Nickle into a new clear container with volume markings.
  13. Let it sit at 4C to allow for all the Nickle to deposit at the bottom of the container.
  14. Aspirate off the RM034 - List of Commonly Used Lab Buffers#20% ETOH|20% ETOH at the top of the bottle without disrupting the Nickle at the bottom.
  15. Resuspend the Nickle with an equal volume of RM034 - List of Commonly Used Lab Buffers#20% ETOH|20% ETOH to create a 1:1 solution.

Next Steps

The regenerated Nickle can be used in more Protein Purifications.

Appendix

Recipe's for Buffers

100mM NiSO4

A reagent used in the process of regenerating Ni-Resin. This buffer should be stored in the cold room.

  • 26.3g of NiSO4 • 6H20 (molecular weight 263 g /mol)
  • Dissolve in a total of 1000mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

Do Not just immediately dissolve the NiSO4 • 6H20 in 1000mL of DI-H20 because that ignores the volume that the Nickle occupies. Dissolve in a smaller volume of DI-H20, and increase the amount of DI-H20 until the total volume reaches 1000mL

50mM EDTA

A reagent used in the process of regenerating Ni-Resin. This buffer can be stored either at room temperature at Eman's Bench or in the cold room.

  • 100mL of 0.5M EDTA
  • 900mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

0.5M NaOH

A reagent used in the process of regenerating Ni-Resin. This buffer can be stored either at room temperature at Eman's Bench or in the cold room.

  • 20g of NaOH
  • Dissolved in 1000mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

Be careful as the reaction for dissolving NaOH in H20 is exothermic so the glass will heat up as you dissolve the NaOH.

20% ETOH

A reagent used to mix with the Nickle Resin to create a slurry suitable for storage. This buffer should be stored in the cold room. Any slurry created from this buffer should also be stored in the cold room.

  • 200mL of 100% ETOH
  • 800mL of DI-H20

Footnotes