His-tag/Ni-NTA purification for secreted proteins derived from Eukaryotic cells

Updated: 11.11.21 KL

This is an overview of a simple His-tag purification for secreted proteins found in the media of mammalian or insect cells.

The principal is identical to that of traditional Ni-NTA. We have only switched the type of resin. Do not use normal Ni-NTA resins. Eukaryotic cell culture media contains EDTA/compounds/proteins that remove the immobilized metal on normal NiNTA resins.

More information on the resin can be found here

Resin: Protein Ark Ni-Advance (https://proteinark.com/index.php?route=product/product&product_id=2277)

or Cytiva Ni Sepharose Excel (https://www.cytivalifesciences.com/en/us/shop/chromatography/resins/affinity-tagged-protein/ni-sepharose-excel-histidine-tagged-protein-purification-resin-p-06009)

Buffers:

Wash Buffer : 1X PBS or any standard buffer ie. 300 mM NaCl, 20 mM HEPES 7.5

Elution Buffer : 1X PBS + 500 mM imidazole, pH 7.5 or 300 mM NaCl, 20 mM HEPES 7.5, 500 mM imidazole 7.5

You will mix the wash and elution buffers in different percentages to make all the appropriate concentrations.

Note: Remember to pH your imidazole. It is also recommended to filter all buffers through a 0.45 um filter at least.

Day 1

Put 1ml (therefore in this case 1 CV = 1 mL) beads for 100ml supernatant
- not always, we just need to have enough to capture all proteins. Beads capacity is assumed to be ~20mg of protein per ml of beads
- we use this amount as it is easy to see and manipulate.
- For larger (1L) cultures we usually use 5 – 10 mL of resin per L.
- Resuspend the beads, put in a tube and wait for it to settle, take out the supernatant (20% EtOH) and wash them with some buffer, let the beads to settle. Remove once again the supernatant and resuspend in buffer. You can use the beads after. This is to remove the EtOH that is used for storage
Add the beads to the S/N and let it agitate overnight in a rolling shaker

Day 2

The next day take the S/N and beads and transfer to a column. For mammalian cells use only plastic columns or glass columns reserved for mammalian cell culture to reduce endotoxin contaimination.
Collect the flowthrough.
Wash beads with 10-20CV of buffer. Collect the wash in a separate container.
Perform step wise washes to remove loosely bound unspecific proteins. However sometimes your protein may also elute. Therefore it is crucial to save all your fractions
- Wash the beads with 5 CV of 90% wash buffer / 10% elution buffer (ie. 50 mM imidazole). Collect liquid that passes through.
- Wash the beads with 5 CV of 80% wash buffer / 20% elution buffer (100 mM imidazole). Collect liquid that passes through.
- Elute the protein with 5 CV of 40% wash buffer / 60% elution buffer (300 mM imidazole). Collect liquid that passes through. Collect each addition of 1 CV in a separate tube if you want to have smaller higher concentration protein.
Elute the protein with 5 CV of 100% elution buffer. Collect the liquid that passes through.

Run a SDS-PAGE gel with reducing buffer.
- Gel sample lanes Flow-through / Supernatant / Washes / Elute
Measure A280 absorbance and note.
Dialysis in desired buffer and leave overnight. Typically 1 – 2 L.

Day 3

Next day remove dialysate and measure absorbance at A280. For general proteins we assume 1 Abs 280 = 1 mg/mL. If you have the sequence you can use the Expasy server protparam to calculate a a more exact conversion factor. Remember to note in your notes which conversion factor you use
Concentrate to appropriate concentration as desired using amicon filters.

Care of beads

Beads can be washed on column with wash buffer and then stored in 20% ethanol. We do not reuse the beads for different projects to prevent containmination until they have been stringently washed.

We typically stringently wash the beads with 1 M NaOH (or supplier’s recommendation), rinse thoroughly with water, then buffer (until neutralized), before storing in 20% ethanol. This can also be done on column.