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Nickle Column Protein Purification

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to purify 500mL - 4L of HEK 293f Supernatant using Nickle Column Chromatography

Reagents and Materials

Things to Note before starting

Determining Column Volume

Column Volume, or the amount of volume the Nickle Resin occupies, determines how much protein the column can absorb. Ideally, the slurry should be stored in a 1:1 ratio of Nickle Resin : 20% ETOH. If this is true, adding 50mL of Slurry should correspond to a column volume (CV) of 25mL. However, this is often inaccurate. A better way to determine CV is to 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.

Location

The entire process takes place in the cold room

Estimated Timing

Nickle Column Chromatogrophy is a very long procedure and can take up to 10 hours to complete 2L of supernatant. While the majority of time is passive, take care to arrange your schedule so that if necessary you can pause the column overnight.

Procedure

Preparing the Protein Supernatant

  1. Move the HEK 293f Cell Culture into 500mL / 1000mL plastic bottles.
  2. Spin the culture media at 4500 rpm for 20 minutes to pellet the cells.
  3. Collect the supernatant into a clean plastic bottle.
  4. Filter the supernatant using 0.65um Filter Paper. 1
  5. Add enough RM034 - List of Commonly Used Lab Buffers#5M NaCl|5M Nacl to the filtered supernatant to bring the final concentration of NaCl to 0.25M.
  6. Add 20-80mL of Ni-Agarose Slurry. 2
  7. Let the supernatant and the Ni-Agarose Slurry incubate in the cold room on the shaker for 1 hour.

Running the Column

  1. Prepare collection flasks of sufficient volume to collect the flow through. 3 Place the collection bottles underneath the column
  2. Add the 250mL Reservoir to the top of the column.
  3. Pour as much supernatant into the top of the column (~300mL) as possible.
  4. Add more supernatant into the column each time the 250mL Reservoir is empty. The supernatant should otherwise remain on the shaker in the cold room to prevent the Ni-Resin from depositing on the bottom of the flask.
  5. After the entirety of the supernatant has gone through the column, measure the height of the Ni Resin to get an accurate CV. The CV should range from 20-50mL. If the CV is greater than 50mL, this process should be split into multiple columns.
  6. Prepare a new flow through collection bottle 3 and place underneath the column.
  7. Pour all the collected Flow Through into the column. Keep refilling the reservoir with Flow Through #1 each time the reservoir empties.
  8. After all the Flow Through #1 has gone through the column, save and label the collected Flow Through #2. It can be stored at 4C for 1 month.
  9. Prepare a new collection bottle3 to collect the Wash and place below the column.
  10. Add 20CVs of RM034 - List of Commonly Used Lab Buffers#1X Wash Buffer|1X Wash Buffer to the column, refilling the reservoir as necessary.
  11. After all the wash buffer has gone through the column, save and label the collected Wash Fraction. It can be stored at 4C for 1 month.
  12. Cap the column.
  13. Add 1.5 CVs of RM034 - List of Commonly Used Lab Buffers#1X Elution Buffer|1X Elution Buffer. Let it incubate for 20 minutes.
  14. Uncap the column and collect the Eluted Protein.
  15. After all the Elution Buffer has gone through the column, re-cap the column.
  16. Add 1.5 CVs of RM034 - List of Commonly Used Lab Buffers#1X Elution Buffer|1X Elution Buffer. Let it incubate for 20 minutes.
  17. Uncap the column and collect the Eluted Protein.
  18. After the Elution Buffer has gone through the column, save and label the collected Protein Elute. It can be stored at 4C for 1 month.

Clean Up

  1. Cap the column
  2. Wash and resuspend the spent Resin with RM034 - List of Commonly Used Lab Buffers#20% ETOH|20% ETOH. Transfer the spent resin to a bottle to be stored until it can be cleaned up and regenerated.

Next Steps

The collected fractions (Flow Through #2, Wash, and Elute) should be concentrated and then can be quantified by the Protein Nano Drop in the Hutchinson Building. The fractions can also be run on an SDS Page Gel and visualized with a Coomassie Stain.

Appendix

Recipe's for Buffers

1X Wash Buffer

The reagent actually used in Nickle Column Chromatograph. This should be stored in the cold room and wrapped in aluminum foil.

  • 55mL of 10X Wash Buffer
  • 500mL of PBS

1X Elution Buffer

The reagent actually used to Elute the Protein during Nickle Column Chromatography. This should be stored in the cold room and wrapped in aluminum foil.

  • 55mL of 10X Elution Buffer
  • 500mL of PBS

Footnotes

  1. We have a re-usable vacuum Filter that utilizes 0.65um Filter Paper. Ask Eman Taha for more information. ↩

  2. The exact amount of Nickle Agarose Slurry you should add depends on the amount of Protein Supernatant you have. On average, the CV is proportional to the half of the Nickle Agarose Slurry. You should be aiming for a CV between 20-50. ↩

  3. The flow through will have the same volume as the initial Protein Supernatant input. The Wash Buffer will have a volume equal to 20CVs. Use the appropriate bottles to collect that volume of material. ↩↩↩