How long do I need to treat transfected HEK 293 cells with Brefeldin A and the concentration need to see endo H resistance in glycosylated protein ?

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Jürgen Denecke

5 micrograms per millilitre Brefeldin A in your cell culture and incubation for 1 hour will be enough to get mixing of Golgi and ER membranes, so your protein will acquire endoH resistance if it is competent for complex glycan formation. I recommend to do it when your protein is well expressed (i.e. 24-48 hours after transfection). If you add Brefeldin A immediately after transfection, expression levels will be reduced.