Mouse IgG1 binding to Protein A Affinity columns is weak. In your experience, what buffer could improve the binding? I read about pH 8.0, 3 M NaCl, some glycerol...
Do you have any suggestions? Also, do you have any suggestions about the better column for this purpose ? We are using HiTrap rProtein A from GE and, in some Ab, the binding is less than 1 mg per ml of resin.
We know we can use Protein G columns but we preffer Protein A in order to elute at mild pH´s.
Thanks!!
This protocol uses a binding buffer consisting of 0.1M Sodium Phosphate, 0.15M NaCl,
pH 7.4.
https://www.kpl.com/docs/techdocs/purifigg.pdf
This protocol uses 1 M potassium phosphate, pH 9.0.
http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/antibody-purification.html
Here is a very detailed protocol:
https://www.weizmann.ac.il/Biological_Services/sites/Biological_Services/files/uploads/protocol5.pdf
Hello! I assume that you are purifying from hybridoma supernatant? The buffer you are using seems correct 0.15M phosphate pH 8.0. I am assuming you meant 300mM NaCl. 3M would be too high. Glycerol is not necessary. Are you eluting with pH 2.0-2.5 glycine? We commonly elute with 0.1 M glycine pH 2.0 into 3M pH 8.0 Tris. Basically put a bunch of microcentrifuge tubes in a rack (or fraction collector if you have one) with 200ul of 3M Tris pH 8.0 and start the elution with the pH 2.0 glycine. Run an SDS-PAGE gel with the fractions or you can even test with Bradford reagent on a strip of parafilm next to the tubes. Just add a drop of eluant to a drop of fresh Bradford reagent and if it turns bright blue there is your protein. It is worthwhile to buffer exchange the antibody containing fractions afterwards. In my experience the 'gentle elution buffer' from Life Technologies only works well with buffer exchange prior to loading the column. The binding buffer is just pH 8.0 phosphate and 150mM NaCl. So you probably don't need to buy the binding buffer from Life Technologies. If you can, prior to loading the Protein A column, buffer exchange the supernatant with tangential flow filtration. You can also buffer exchange with a centrifugal concentrator and then use dialysis tubing. If you plan on purifying many anitbodies or if this is a mainstay of your lab. It is worthwhile to invest in a TFF system. The one we use is from Millipore and uses a 10 kDa cutoff. I put a link in the answer. There are some single use options which might be better/cheaper. Once you buffer exchange and reduce the volume to ~ 200ml. It makes it much easier to run over the Protein A column, which you could do a few times. However I bet you would max out the binding capacity once buffer exchanged.
http://www.emdmillipore.com/US/en/product/Prep%2FScale-Spiral-Wound-Ultrafiltration-Modules,MM_NF-C610
Hello Juan,
Mouse IgG1 has very low affinity for protein A and you won't be able to obtain any significant increase in binding by buffer changes. Your best bet is to use Protein G. Your concern about elution conditions is legitimate but remember that IgG1 also has lower affinity for protein G than other IgG subtypes so you will be able to elute with milder conditions.
Test a range of conditions (pH5-2) and select the mildest condition that results in elution of most or all of your antibody. Drop the elutions directly into tubes containing high pH buffer (pH9.5 to 10) to neutralise rapidly and minimise the acid-exposure time.
Good luck.
Well I would be careful about eluting with pH 5 into pH 10 to neutralize unless the concentration and volume keep you away from high pH because it can cause aggregation of your antibody. Buffer exchange will increase the binding to to your column, especially if you use hybridoma SFM media w/ glutamax. It also kind of a drag to pump massive amounts of hybridoma supernatant over a protein A column (usually overnight) at 0.3 ml/min. If you can't buy a TFF, or don't need one, you can use a centrifugal concentrator that will do 100ml of supernatant at a time. Buffer exchange into binding buffer then you can concentrate down to 100ml or less and then with your low affinity IgG1 run it over the protein A column several times to get out your weakly binding antibody. Good Luck!
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html
Dear Juan
It is demonstrated that antibodies binding to protein A is realized, at neutral or alkaline pH values, using hydrophobic interactions involving a conserved histidine residue located in the protein A binding site of IgG. Then IgG releasing from immobilized protein A is commonly achieved by lowering the pH using an acidic buffer.
For more informations please check following papers:
-Duhamel RC et al., pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-sepharose, J Immunol Methods 31(3–4), 211 (1979).
-Gagnon P (1996). Purification tools for monoclonal antibodies (Tucson: Validated Biosystems Inc).
-Schwartz L (1990), In bacterial immunoglobulin-binding proteins, Vol. 2, M. Boyle, ed. (San Diego: Academic Press), p. 309.
Hoping to contribute
Marius
Mouse IgG1 will bind pretty well to Protein A at pH 8.5-9. Hybridoma culture supernatants can be adjusted to pH 9 using Trizma or other buffer (using the phenol red colour as a guide). Some protocols suggest high salt , 1-2M NaCl, will enhance affinity as well; Binding Buffer A (1.5 M Glycine/NaOH buffer, 3 M NaCl, pH 9.0)
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