UniGlow™ is ready-to-use and no mixing is required. UniGlow™- One Component Chemiluminescent Substrate is for use in microwell or membrane applications. Protect from light - UniGlow™ Substrate is a highly sensitive detection reagent. Equilibrate to room temperature before use and aliquot into a clean container. Use ~100µL/cm2 of membrane. Optimal detection visualized after contact of substrate with HRP enzyme on membrane for ~1-10min. Blot off excess substrate before imaging. Always carefully optimize all components of individual assays (antigens, antibodies, conjugates…) to minimize background reactivity associated with non-specific binding.