Sigma Aldrich
CDP-Star Chemiluminescent Substrate
C0712-100ML
Info: Supplier Page
Hedgehog signaling plays a critical role in embryonic development. It also controls adult tissue homeostasis and deregulation of this pathway results in basal cell carcinoma and medulloblastoma. A unique feature of this pathway is that it requires a tiny antenna-like organelle called the primary cilium for signal transduction. My goal is to understand the molecular mechanism of hedgehog signal transduction at the primary cilium. This involves detecting protein-protein interactions of the pathway components, analysis of localization patterns during signal transduction, and functional assays that measure pathway activity. In this experiment, the effect of depleting Smoothened (a GPCR signal transducer) on pathway activity was analyzed in C3H10T1/2 cells after ligand treatment.
Luminescence measurement in 96-well format.
C3H10T1/2 cells
None.
1. C3H10T1/2 cells seeded in a 96-well dish were transfected with control siRNA or Smo siRNA.2. 48 hours later, ligand treatment was done for 36 hours. This results in differentiation of these cells and express alkaline phosphatase.3. Remove media and rinse with 1x PBS (100µl per well).4. Lyse in 50µl lysis buffer per well (Lysis buffer: 100mM tris pH 9.5, 250mM NaCl, 25mM MgCl2, 1% TritonX-100); incubate 45min at RT on the shaker.5. Transfer 10µl of lysate to 96-well plate (white wall) and add 50µl of room-temperature CDP-STAR reagent per well, incubate for 15min at RT in the dark (cover with aluminum foil).6. Read plate in a plate reader using settings optimal for luminescence measurement.
N/A
Easy to use reagent and works well with frozen lysates.
Good option for high-throughput assays.