I am performing IgG purification and I have to show my results on SDS-PAGE. I use 10% tris glycine gel and prepare the samples under non reducing conditions. I am new to antibodies and therefore need some help. First of all, my main band in cell lysate is about 50 kDa for some reasons. Purified antibody showed the same band and also one at around 250kDa.
Initially, I did reducing conditions and got one extra band at around 25 kDa in elute, so I changed to non-reducing. I attached SDS-PAGE gel (non-reducing), L- lysate, E- elute, M- marker (10-250 kDa)
On a non-reducing gel, an IgG should run at 150 kDa. On a reducing gel, you should get bands at 50 and 25 kDa (heavy and light chains, respectively). The main band in your lysate lanes may be serum albumin (66 kDa) if there was serum involved in production of your antibody.
Adam B Shapiro Thank you, I am not sure if serum albumin was involved in production, I only know that they used 293F cells. if It is serum albumin, the question arises, why is it being purified as the main band, I use generic antibody binding protein. Its similar to protein A, but slightly modified.
The concentration of albumin in culture media containing serum is quite high. There are other proteins, too, but albumin is by far the most abundant. It can be difficult to get rid of all of it when purifying IgG from culture medium. There are resins available to help remove it, or you may be able to adapt the cells to grow in serum-free medium, which lacks albumin.
You mentioned a lysate, suggesting that the IgG was produced within the cells rather than excreted into the medium. If this is the method of production, thoroughly washing the cells to remove external albumin before preparing the lysate would probably help. Also, make sure there is no albumin in the lysis reagent, of course.
Adam B Shapiro thanks a lot. I will try my best to get rid of it. Actually the idea of my work is to purify IgG, Fab and scFv using a new purification system. I was just confused when I observed not related bands in my elute as albumin is not supposed to have any interaction with my purification media. Probably a few extra washing steps can solve this problem. Thank you again for clarifying all the aspect.
You can try recombinant protein G which has been engineered to remove the albumin binding site that is present in both native Protein A and Protein G. I purify MAb IgG from liters of media containing IgG-depleted fetal calf serum without significant albumin contamination.
https://www.cytivalifesciences.com/en/us/shop/chromatography/resins/immunoprecipitation/protein-g-sepharose-4-fast-flow-antibody-purification-resin-p-00067
Mary Cristine Charlesworth Thank you for your answer, however I am testing a new purification method and therefore can't try comercially avaliable antibody binding proteins. I already solved this problem by adding more washing steps. Instrestingly some of IgG still got reduced into heavy and light chains even under non-reducing conditions. And this is a problem I am trying to solve now.
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