ECL for Westerns - Homemade

[Luke]

Homemade ECL

 
Solution 1.                  Solution 2.         

100uL 250mM luminol            6uL H2O2, 30%
44uL 90mM p-coumaric acid            1mL 1M Tris-HCl, pH 8.5
1mL 1M Tris-HCl, pH 8.5            9mL ddH2O
8.85mL ddH2O

1.   Prepare solutions fresh right before ready to use
2.   All other steps in western are performed exactly as you normally would
3.   Combine solutions and mix.
4.   Add blot and incubate 1 minute
5.   Blot up excess substrate and develop as usual
6.   This ECL is very sensitive and avoid having excess on blot.


Luminol= 3-aminophthalhydrazide (Fluka): 0.44g into 10mL DMSO, aliquot

p-coumaric acid: 0.15g into 10mL DMSO, aliquot