Chemiluminescence Using HRP

After incubation with antibody and second antibody HRP-conju-gate, wash the blot three times with about 1 ml/cm of Soln. A for 5 min each. Be sure that no buffer used after the incubation with conjugate contains sodium azide or another inhibitor of HRP. 

Pour off the washing buffer and add ECL reagent mix (1 vol. reagent 1 and 1 vol. reagent 2, about 0.5 ml/cm ) and incubate for 30 s to 1 min at RT. Remove membrane, drain, and place face down to a thin clear plastic wrap. Form a moisture-tight envelope from the remaining wrap and place the wrapped membrane with blot face down onto an X-ray film in a dark room. Be sure that no liquid contacts the film. 

The exposure time ranges from 2 to 30 min. An alternative to X-ray film is a digital camera. 

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