Chemiluminescent Detection

Contributed by Ian Chin-Sang, Queens University, ON, Canada

Homemade chemiluminescent detection recipe

Requirements

p-coumaric acid (Sigma #9008) 3-aminophtalhydrazide (Luminol, Sigma #09253) 1 M Tris pH 8.5 30 % H2O2 90mM p-coumaric acid in DMSO (store at -20°C) 250 mM Luminol in DMSO (store at -20°C)

Method

  • Solution A (50ml):
  • 5 ml 1M Tris pH 8.5
  • 45 ml water
  • 110 ul 90 mM coumaric acid (0.2mM coumaric acid)
  • 250 ul 250 mM Luminol (final 1.25mM Luminol)

Keep 2-3 weeks at 4°C

  • Solution B (1ml):
  • 100 ul 30% H2O2
  • 900 ul water

Keep 2-3 weeks at 4°C

  • Combine 1 ml Solution A + 3 ul Solution B (5ml Solution A +15ul Solution B for each blot)

This method is based, with permission, on an original protocol available here.