I try to purify a membrane protein with a strep-tactin column. However, the protein doesn't elute after the addidition of dethiobiotin 2.5 mM but "elutes" during the regénération (with NaOH 0.5 M).
(detergent used DDM : 0.05% that is above CMC, Tris 100 mM, 1 mM EDTA and 150 mM NaCl)
I think that the protein could precipate in the column.
Do you have other possible explanations ? Do you have other suggestions ? change the NaCl concentration (I used 150 mM here) or increase the desthiobiotin concentration could be good ideas ? If you have suggestions thank you.
Hi there,
If specific competitor is unefficient it means the interaction between your protein of interest and the matrix is not the one you were expecting. On column aggregation is very likely in your case... Did you equilibrate the column with the DDM containing buffer before loading the protein sample? And did you maintain its concentration all along the purification steps?
Thanks for your reply.
Yes I equilibrated, loaded, washed and eluted with the same DDM concentration (0.05 %).
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