Anyone have experience with poor binding of Fc tagged proteins to protein A columns? We have a mouse IgG2a Fc tagged protein that is not binding well to either a protein A or protein G column.
I use the new rProtein A fastflow sepharose from GE health, I collects conditioned media which contains fusion protein secreted form 293T cells. Then I did the purification and collected each fraction of every purification step. Every time there are nearly 20% protein which can bind to the beads. I tried many different methods, and I added iodoacetamide and DTT to the condition media to reduce disulfide bond. But still no help to the binding.Now I am thinking to partially denature the protein with Urea. Do you have a protocol about when and how to add urea to the fusion protein?
Hi Xia
Check the pH of the cell culture supernatant and of course, total and dynamic capacity of your Protein A column. IgG2a shows good affinity for protein A and does not needs any special conditions for binding. Neutral pH and flow rate around 1-2 ml/min is a good start point. The binding capacity of the protein A for IgG2a is more than 10mg/ml gel, so be careful do not exceeding the total capacity of your column. If you reduce to 50 ml the sample volume before apply into the column, try to perform a desalting vs binding buffer. It may eliminate some small peptides or interfering substances.
Hi Xia
Desalting is type of size exclusion chromatography, where only molecules of MW smaller than 5 KDa are included into the gel. So, in this sense you can separate salts, small peptides and other molecules (< 5 KDa) from proteins. The more popular medium for this purpose is Sephadex G 25 with exclusion size of 5 KDa. Other gels with differents "cut off" are available. The result obtained by this method is similar to dialysis but is faster and secure. There are prepacked columns like PD-10 or HiPrep 26/10 from GE for small and "large" sample volume. If you can not do chromatography, I suggest you ultrafiltration method using a membrane with cut off as higher as possible. Then, you can concentrate and wash your sample with the appropriate buffer to affinity chromatography on Protein A
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