Which technique is used to determine whether a protein is GPI-anchored?
Distinguishing GPI anchoring from transmembrane helices or other lipid modifications requires an enzymatic strategy that specifically targets the glycolipid linkage chemistry. A mature GPI anchor contains phosphoethanolamine in amide bond to the protein C-terminus, a conserved tetrasaccharide core of three mannose residues and glucosamine, and phosphatidylinositol whose diacylglycerol or alkylacyl side chains embed in the exoplasmic leaflet. Bacterial phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis cleaves the phosphodiester between glycerol and phosphate, releasing protein-glycan fragment soluble in aqueous phase. After treatment, membrane extracts are subjected to Triton X-114 phase separation or high-speed centrifugation; authentic GPI proteins shift from detergent-rich pellet to aqueous supernatant detectable by immunoblotting while transmembrane helices remain membrane-bound. Resistance to hydroxylamine and sensitivity controls with known GPI proteins like alkaline phosphatase, Thy-1, CD14, CD55, CD59 and prion protein validate specificity. This assay remains standard for confirming outer leaflet glycolipid tethering in trafficking and raft studies. Combined detection with anti-CRD antibodies recognizing cross-reacting determinant after cleavage further validates GPI identity and distinguishes from transmembrane contamination.
Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 10, GPI-anchored proteins and PI-PLC assay.