The Home-made ECL Western blotting detection reagents

Stock solutions

250 mM Luminol (5-amino-2,3-dihydro-1,4-Phthalazinedione)

’Sigma’, A-8511, Fw 177.2 g/mol:

443 mg/10 ml DMSO, store the aliquots of 50 l at -20C.

90 mM p-Coumaric acid (4-Hydroxycinnamic acid)

’Sigma’, C-9008, Fw 164.2 g/mol:

74 mg/5 ml DMSO, store the aliquots of 22 l at -20C.

1 M Tris/HCl, pH 8.5

’Sigma’, T-1378, Fw 121.1 g/mol:

12.11 g/100 ml H₂O

30 H₂O₂

Solution A (keep in a dark bottle)

9 ml H₂O

1 ml 1 M Tris/HCl, pH 8.5 (0.1 M final)

22 l 90 mM p-Coumaric acid (0.196 mM final)

50 l 250 mM Luminol (1.24 mM final)

Solution B (keep in a dark Eppendorf tube)

900 l H₂O

100 l 30 H₂O₂ (3 final)

The solutions are stable for 2-3 weeks at +4C

Mix 1 ml of solution A with 3l of solution B (H₂O₂ 0.009, Luminol 1.24 mM, and p-Coumaric acid 0.196 mM final).

The final volume of detection reagent required is about 0.1 ml/cm².

It isn’t good for exposure time longer than 30 min. For shorter time efficiency is even better that with commercial ECL.

2003 07 17 – Oliver & Julius