After separating membrane proteins from the membrane environment, we are seeking robust and efficient ways to exchange detergents for X-ray scattering structural experiments. Are sizing columns or desalting columns reasonable choices or are there good alternatives?
Hi John, excellent question! Complete detergent exchange is difficult and the succes depends on the exact detergents you are exchanging. I have exchanged DDM for a number of different detergents by washing immobilised proteins extensively (immobilised with affinity- or other chromatography) with a buffer containing the detergent of choice. Final polishing can be done with Size Exclusion Chromatography, provided that the micells of the detergent that you want to remove can be well separated from the protein-detergent complex. Alternatively, you could consider dialysis (will only work for detergents with a high CMC) or strongly diluting your protein in a buffer with the desired detergent, followed by a concentration step. Keep in mind that when you concentrate the protein, you might end up also concentrating the detergent if the MWCO of your concentrator is lower than the micell's molecular weight.
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