Application of a Core Streptavidin Stv13 Fused CpOGAD298N in Protein O-GlcNAcylation Detection
Oxygen-linked β-N-acetylglucosamine(O-GlcNAc)modification is a dynamic,reversible post-translational modification that occurs on the hydroxyl group of protein serine and threonine residues and plays important roles in many key cellular processes.Antibodies are commonly used in the detection of protein O-GlcNAcylation,but their specificity against O-GlcNAcylation or the molecular weight range of detected proteins remains to be improved.The Clostridium perfringens OGA mutant(CpOGAD298N)has been applied in the detection of O-GlcNAcylation in a far-Western blot(Far-WB),due to its advantage in the binding with O-GlcNAc.In this study,CpOGAD298N was fused with a core streptavidin Stv13.Based on the specific binding property between biotin and streptavidin,we established a fast O-GlcNAc Far-WB assay method,which is verified using short OGT(sOGT),de-O-GlcNAc sOGT,cell lysates and HNF1A proteins.Taken together,our work provides a specific and time-saving Far-WB method(5-7 hours),which can be effectively used in the detection of protein O-GlcNAcylation.
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