I had a lab where I had to do western blot to detect b-galactosidase through che

Question

I had a lab where I had to do western blot to detect b-galactosidase through chemilumescence. A SNAP id system was used to shorten the western blotting procedure. We used primary and secondary antibodies. The enzyme label on the secondary antibody was HRP (horse radish peroxidase), and the substrate was luminol- a solution of luminol and peroxide was added to the membrane at the end of the western blot so that a chemilumescence reaction would occur and a photon was supposed to be released. The emission was supposed to be detected using a Kodak Imager but nothing showed up. Why might this be? I was thinking maybe the antibodies did not incubate long enough on the membrane (even though I followed the length of time stated in the procedure), or maybe the photon emission decayed because I had to wait about 15-20 minutes after adding the luminol and peroxide before the membrane could be placed in the Kodak Imager (the container that the membrane was in was covered with aluminum foil though to prevent exposure to light in the room). But I thought that adding a peroxide would extend the time of photon emission but i don't know by how much. Any ideas as to what may have happened?

Explanation / Answer

The photon is released but this is low intensity light emission only. So it is not seen for long. The light emission may have died down by the time you placed the membrane in the Kodak imager. For a more intense prolonged and stable emission, you could add para-iodophenol which enables you to have this light emission for long.

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