Hi everyone and expert glycobiologists!
I'm trying to optimize an assay that uses PNGaseF (N-glycosidase; New England Biolabs P0705S) to deglycosylate my cells (Jurkat T-cells). However, I cannot denature the cells (with the provided glycoprtotein denaturation buffer) to conduct this deglycosylation, as I need the live/intact cells for flow cytometry.
I was wondering what range of PNGase F enzyme units + the duration should I begin testing?
Moreover, would I be able to use PBS in place of the glycobuffer 2 given that it is the same pH to conduct the glycosidase reaction?
I see the denaturation reactions usually use 500 U of enzyme but I'm not sure how much I should increase this amount for use on the live cells (in non-denaturing conditions). If it helps, my protein of interest is expressed on the cell surface (membrane bound) and has 4 N-glycans that I would like to remove (in addition to the global deglycosylation from PNGaseF).
Any insights would be much appreciated! :)
Shifa
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