title: Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.
abstract: We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.
authors: Department of Cell Biology, University of Texas Southwestern Medical Center, NL07.138B, 5323 Harry Hines Blvd., Dallas, TX, 75390-9039, USA.
authors: Rubina
authors: R
authors: Tuladhar
authors: Department of Cell Biology, University of Texas Southwestern Medical Center, NL07.138B, 5323 Harry Hines Blvd., Dallas, TX, 75390-9039, USA.
authors: Nageswari
authors: N
authors: Yarravarapu
authors: Department of Cell Biology, University of Texas Southwestern Medical Center, NL07.138B, 5323 Harry Hines Blvd., Dallas, TX, 75390-9039, USA. Lawrence.Lum@UTSouthwestern.edu.
authors: Lawrence
authors: L
authors: Lum
mesh_terms: D000215:Acylation; D000480:Alkynes / Q000737:chemistry; D002384:Catalysis; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D054878:Lipoylation; D051153:Wnt Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D000480:Alkynes; D051153:Wnt Proteins; D003300:Copper
keywords: Biotinylation; Click chemistry; IgG fusion protein; Palmitoleate; Wnt acylation