Small Scale Protein A/G Purification

Updated: 11.11.21 KL

This is an overview of a simple Protein A or G affinity purification. This can be used for purification of antibodies or Fc containing recombinant proteins typically from HEK or CHO cell culture media.

Protein A and G are staphylococcal derived proteins that were found to bind the Fc regions of antibodies. They have different specificities depending on species and isotype.

Fun Background:https://www.cytivalifesciences.com/en/us/news-center/history-of-protein-a-10001

Specificities:https://www.sigmaaldrich.com/CH/en/technical-documents/technical-article/protein-biology/protein-pulldown/protein-a-g-binding?gclid=CjwKCAiAm7OMBhAQEiwArvGi3KYKvLhDM9PA5uuQPfefOSfEdpjCVn69Q7LPCI4SRlvhf2Lb3WPydhoCPhgQAvD_BwE

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 PBS, let the beads to settle. Remove once again the supernatant and resuspend in PBS. 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 20CV of PBS. Collect the wash in a separate container.
• Elute with 5CV 0.1 M Glycine pH 3. Collect elute in a separate container.
• Add 1.2 CV of 1.5 M TRIS pH 8 to neutralize the elute (one could also add the necessary amounts in the elute container and let the elute drip in to neutralize immediately) 
• Run a SDS-PAGE gel with reducing buffer. 
  • Gel sample lanes Flow through / Supernatant / Wash / Elute 
• Measure A280 absorbance and note.
• Dialysis in PBS 1x and leave overnight. Typically 1 – 2 L.

Day 3

• Next day remove dialysate and measure absorbance at A280. For antibodies we assume 1.4 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. An antibody is around 150 kDa.

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 50 M NaOH (or supplier’s recommendation for MabSelect), rinse thoroughly with water, then buffer (until neutralized), before storing in 20% ethanol. This can also be done on column. Contact times may vary. See guide below. https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13118