I want to purify a mouse Fc-tagged fusion protein from culture supernatant, but mIgG1 has a very low-affinity to both Protein A, and with protein G, bovine IgG will interfere with the purification in culture supernatant, making it difficult to purify. Does anyone have an experience with the purification of mIgG using these purification methods? Otherwise can anyone advise me on any other alternative method of purification?
Hi Vishal, in my experience mouse IgG1 can be easily purified with ProteinG or ProteinA. with proteinG you should check if you're using type II or type III (better), while for ProteinA (lower affinity) you can increase the efficiency by checking (and eventually moving) the pH (it should be around 8.0). I suggest to perform a small scale binding trial (immunoprecipitation of your supernatant with both resins, separately, then sds-page run and coomassie) to define their binding capacity, and to decide how to proceed (ProteinA should be cheaper). in my opinion your problem is mainly due to the very high amount of cow IgG present in complete medium or FBS. the quicker way is to work with IgG-free (cow) media (cells adaptation to serum-free media is not your case, mainly indicated to stable cell lines like hybridomas for monoclonal antibody production), or (cheaper way) you could deplete these IgGs (cow) from the standard FBS to be used for cell culture, using the same kind of resin (proteinG o ProteinA) you will use for the purification of your Fc-tagged protein (this procedure requires a bit of attention, just to avoid contaminations in the next cell culture step). I can add more details, if needed. best. Giuseppe
Thanks Giuseppe for sharing your experience. I was thinking of using serum-free media but then I realized that to produce enough amount of protein, I will have to leave the cells in the medium at-least 36-48 hours post-transfection. In this time period, my cells will die due to serum starvation.
I think your second suggestion is useful, as FCS has low levels of antibodies, I would be able to separate them without using lot of beads. Have you depleted IgGs from FCS before? If I use 50ml of FCS, do you know how much ProteinG beads I should use?
usually I deplete 200ml of FBS SA with a 5ml column of ProteinA (hitrap mabselect sure from GE). I perform that on a akta explorer, with a very low flow-rate, over-night at +4C...
once I saved and quantified the stripped cow IgG, and the yield was around 20mg.
Thanks Giuseppe! Could you please give me the catalogue number? there are quite few options available and I am not sure which one to choose
- Badges
- Science method