An affordable way to reveal peroxidase activity without expensive kits...

 

            Protein detection by Western-blot is often performed by revealing peroxidase activity of

            secondary antibodies.

 

            For that one uses ECL or other equivalent reagent, which are quite expensive.

 

            Here is so the recipe of a "home-made ECL":

 

            1) Prepare the following stock solutions:

 

             Luminol

                                                  250mM in DMSO, keep in dark at -20°C

                                                  ex: Fluka, cat. no. 09253

 

 

             Coumaric acid

                                                 90mM in DMSO, keep in dark at -20°C

                                                 ex: Sigma, cat. no. C-9008

 

 

             Tris-HCL

                                                 1 M, pH 8.5

                                                Hydrogen perox. 30%

                              

 

 

            2) Prepare the following solutions:

 

            Solution 1:   (  100ml  )

                                                1 ml luminol 

           

                                                0.44 ml coumaric acid

            

                                                10 ml Tris 1M pH 8.5

            

                                                distilled water to 100 ml final volume

            

                        

 

 

                                              

 

            Solution 2:  (  100ml  )

                                                       64 ul hydrogen peroxide

           

                                                      10 ml Tris 1M pH 8.5

            

                                                       distilled water to 100 ml final volume

 

 

            Store these two solutions in fridge, wrapped in aluminium foil for about 2 months, after

            which sensitivity may decay.

 

            Reveal your western-blots with a 1:1 mix of solution 1 and 2 as you would do with

            commercial ECL reagent.