Homemade ECL

Chemiluminescent Detection Recipe:

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)

Fresh solutions:
2-3 weeks at 4°C

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)

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

To use, mix:
1 ml Solution A + 3 ul Solution B 
(5ml Solution A  +15ul Solution B for each blot)

Another Protocol from :

We use a chemiluminescent reagent mix described by Yakunin and Hallenbeck:

Yakunin AF and Hallenbeck PC. (1998) 'A luminol/iodophenol chemiluminescent detection system for western immunoblots'.  Analytical Biochemistry 258, 146-149.

The reagents are simple to prepare and cost a fraction of commercial preparations.

Solution A

100mM glycine pH 10 (with NaOH)

Solution B

0.12% (w/w) hydrogen peroxide in water

Storage  Solution A needs to be kept in the dark.  Both solutions will keep at 4oC for at least a year.

Use  Mix equal volumes of solution A and solution B.  Incubate with blot for 1 minute.  Drain excess liquid and wrap blot in plastic film to prevent it drying out.  Expose to film.