User Instruction Store reagents A and B in the darkness at 4-8°C.Mix equal volumes of reagent A and B (chemiluminescent substrate) in a clean container and equlibrate to room temperature 30 minutes before use.Prepare your membrane prior addition of chemiluminescent substrate, by a wash with a buffer used in your protocol (PBS or TBS or TBST-T). This will allow to remove any background prior to substrate contact.Optimal visualization is obtained up to 20 minutes after substrate contact. Incubation for 2 to 5 minutes is usually enough.Remove excess substrate by filter paper.Cover blot with clear plastic wrap or sheet protector and expose either with x-ray film or CCD camera.In some cases Tween can quench the reaction. For best results clean containers and high quality water has to be used. HS code for this product is: 3822.00.0002.