Enhanced detection of in vivo SUMO conjugation by Ubc9 fusion-dependent sumoylation (UFDS).
The bottleneck in studying protein sumoylation-the conjugation of the small ubiquitin-like modifier (SUMO)-is the detection of the low level of in vivo sumoylated proteins. The Ubc9 fusion-directed sumoylation (UFDS) system strongly enhances the in vivo sumoylation of a substrate protein at its specific sumoylation site. UFDS utilizes an expression plasmid for the protein of interest fused to the SUMO-conjugating enzyme Ubc9. When expressed in HEK293, COS-7, HeLa, or CHO cells, the fused target protein is conjugated with endogenous or coexpressed SUMO at its native sumoylation sites. This sumoylation requires neither SUMO ligase nor any extracellular stimulation and is easily detectable by fusion protein- or Ubc9-specific Western blotting with commercially available antibodies.
Niedenthal
Rainer
R
Institute of Biochemistry/Physiological Chemistry, Medical School Hannover, Germany.
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D006224:Cricetinae; D004526:Efficiency; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9