Home-made ECL Chemiluminescent Reagents

Stock solutions:

250mM 3-aminophthalhydrazide (luminol): Sigma #A-8511

0.44g/ 10ml DMSO (store –20 1 year)

90mM p-coumaric acid: Sigma #C-9008

0.15g/10ml DMSO (store –20 1 year)

30% H2O2 Sigma #H-0904 (100ml)

100mM Tris, pH 8.6 (need to be very accurate)

ECL stock solutions:

Solution A:

2ml 250mM luminol

440ml 90mM p-coumaric acid

100ml 100mM Tris, pH 8.6

Solution B:

120ml 30% H2O2

100ml 100mM Tris, pH 8.6

Keep at 4 C, mix 1:1 before use.

**The original protocol calls for making Solutions 1 and 2 fresh each time. We typically keep stock Solutions 1 and 2 premade for up to one month and have not noticed loss of activity.

**Once the blot has been incubated in the Solution A and B mixture the signal tends to decay faster than commercially available ECL

500 mL 100mM Tris pH 8.6

6.1g Tris base

400mL ddH2O

pH to 8.6 with 0.5mL 10M, 2mL 1M HCl

ddH2O to 500 and recheck pH

autoclave and store at RT