I have expressed an Fc fusion protein in 293F cells, and the expression in the supernatant was so good, but when I purified it with Protein G agarose and Protein A agarose, it cannot be eluted. I have used different buffer(such as 0.1 M Glycine, pH2.8 and 0.1 M Glycine, pH10.0 ) to elute, but both of them didn't work. How can I purify this protein? Thanks!
How did you treat the sample before loading? Did you filter it, and if so is it possible the protein adsorbed to the filter. If you did not purify the harvest is it possible some lipids are clogging the column? Furthermore, what are the process parameters, was the column equilibrated properly? Have you determined the protein pH and salt stability, perhaps it is inactive or unstable in the elution buffer.
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