Smoothened (SMO) is a GPCR-like receptor which, with Patched, mediates hedgehog signaling to regulate gene expression through the Gli transcription factors. 20(S)-hydroxy Cholesterol (20(S)-OHC) is an oxysterol which binds SMO and activates hedgehog signaling (EC50 = 3 μM), and this activation is selective for the nat-20(S)-OHC enantiomer. Nat-20(S)-OHC synergizes with the SMO agonist SAG, suggesting an allosteric effect. Nat-20(S)-yne is a form of nat-20(S)-OHC with a terminal alkyne group, which can be used in linking reactions known as click chemistry. Click chemistry involves highly dependable and specific azide-alkyne bioconjugation reactions and can be used to capture or immobilize bioactive molecules. Thus, nat-20(S)-yne has been conjugated with magnetic beads to demonstrate that nat-20(S)-OHC directly binds SMO.