Western Enhanced Chemiluminescent (ECL) Substrate (Low Pico to Low Femto Sensitivity)

Powder Dissolution
1. To two 5 L acid and alkali resistant bottles. Take an amber bottle for luminol solution, and a normal bottle for peroxide solution.
2. Unpack the bag and pour all the powder in each bottle.
3. Add deionized water to make a volume of 5 L.

1. Keep the membrane moist in the wash buffer while preparing the substrate mixture. Please ensure the membrane does not dry out during the subsequent steps.
2. Mix Luminol solution and Peroxide Solution in a 1:1 ratio, and thoroughly mix the chemiluminescent substrate solution well.
3. Prepare 0.1 ml of solution per cm² of membrane. For a mini-sized membrane (7 × 8.5 cm), 5 ml of solution is sufficient. For a midi-sized membrane (8.5 × 13.5 cm), 10 ml of solution is sufficient.
4. Place the membrane with the protein side up on a clear and level surface or in a clean container.
5. Remove the membrane from the chemiluminescent substrate solution and drain off excessive solution.
6. Place the membrane in a plastic sheet protector or in plastic wrap to prevent the membrane from drying.
7. Image the membrane with a digital imager or by exposure to X-ray film.