Wnt3a requires a hydrophobic carrier to maintain its biological activity.

<p>To generate “Wnt3a+CHAPS” (red), 30 ng/µL of Wnt3a protein was diluted in a buffer containing 1× PBS, 0.5 M NaCl, and 1% CHAPS yielding a final CHAPS concentration of 1% and final Wnt3a concentration of 1.95 ng/µL. To generate “Wnt3a−CHAPS” (green), 30 ng/µL Wnt3a protein was diluted in a buffer containing 1× PBS and 0.5 M NaCl yielding a final CHAPS concentration of 0.004% and final Wnt3a concentration of 1.95 ng/µL. Aliquots of each solution were incubated at 23°C for the indicated times, then applied to LSL cells (see Methods). The output of the LSL assay is luciferase activity/beta galactosidase activity (i.e., luc/lac); percent activity is defined as % activity = , where 100% activity = 0.05 ng/µL Wnt3a. (A) After 24 h at room temperature, Wnt3a+CHAPS retains of its 98.0% activity (red line) whereas Wnt3a−CHAPS (green line) retains 11.4% of its activity. (B) Anti-Wnt3a immunoblotting confirms that equivalent amounts of Wnt3a protein were present in both +CHAPS and −CHAPS conditions at the beginning (0 h) and conclusion (24 h) of the incubation period. (C) Immunoblot analysis confirms that in the presence of CHAPS Wnt co-fractionates with lower density sucrose fractions (#1–3). In the absence of CHAPS the protein precipitates as the majority of the protein is found in a high density fraction (lower panel). (D) Sucrose density gradient centrifugation and Wnt reporter assay, demonstrate that in the presence of CHAPS, Wnt activity (red line) migrate to lower density fractions. Without CHAPS, Wnt activity is not detected (green line) (E) To generate “Wnt3a+lipids” (blue), 30 ng/µL of Wnt3a protein was diluted in DMPC∶cholesterol 90∶10 lipid mixture containing 1× PBS yielding a final lipid concentration of 14 µmoles and final Wnt3a concentration of 1.95 ng/µL. To generate “Wnt3a−lipids” (green), 30 ng/µL Wnt3a protein was diluted in a buffer containing 1× PBS and 0.5 M NaCl yielding a final CHAPS concentration of 0.004% and final Wnt3a concentration of 1.95 ng/µL. Both the samples were incubated at room temperature for 24 h. (F) Anti-Wnt3a immunoblotting confirms that equivalent amounts of Wnt3a protein were present in both +lipids and −lipids conditions at the beginning (0 h) and conclusion (24 h) of the incubation period. Data are mean ±SEM from, or are representative of, at least three independent replicates. (G) Wnt3a incubated in the presence of CHAPS (red bar) and lipids (blue bar) for 6 h at room temperature. Wnt3a samples were prepared as described in (A) and (E). (H) Wnt3a incubated in the presence of CHAPS (red bar) and lipids (blue bar) for 6 h at 37°C.