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This video tutorial demonstrates how to prepare a Western blot membrane for chemiluminescent detection. After antibody incubation and washing, the membrane is ready for enhanced chemiluminescent (ECL) substrate. Use 7ml of substrate for mini-sized membranes and 12ml for medium-sized for optimal results. A clean sheet protector reduces substrate volume needed and is recommended. Incubate for five minutes to ensure uniform coating, displacing wash buffer. Sheet protectors are inexpensive and disposable, and membranes can be kept inside for imaging.