Detection and analysis of proteins modified by O-linked N-acetylglucosamine.
First, a protocol for increasing the stoichiometry of O-GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O-GlcNAc-modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O-linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O-GlcNAc modified proteins, including labeling of O-GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O-GlcNAc transferase and O-GlcNAcase activity, respectively.
Zachara
N E
NE
The Johns Hopkins University, Medical School, Baltimore, Maryland, USA.
Cole
R N
RN
Hart
G W
GW
Gao
Y
Y
D000117:Acetylglucosamine / Q000737:chemistry*; D002846:Chromatography, Affinity; D015151:Immunoblotting; D037102:Lectins / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification
D037102:Lectins; D011506:Proteins; D000117:Acetylglucosamine