Elispot Discussion

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Elispot Discussion

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Elispot Discussion: Please, feel free to start discussions about the Elispot technique, methods, ideas, questions, experience etc. With your input, we would like to make this website to a special interactive place for everyone who is interested in the Elispot assay.

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Subject Substrate

Date

Author Mike (mikec@zellnet.com)



Hi, Karen; we have performed many assays comparing the sensitivity of different substrates. If you use AP+BCIP/NBT, you should titrate the activity of your enzyme. Otherwise, you might end up with overdeveloped areas. BCIP/NBT is pretty much comparable with the sensitivity of AEC and DAB, substrates for the HRP. DAB is carcinogenic, so I do not recommend to use it. We have a protocol on our web site for AEC. Another substrate for HRP is Novared. It is a more concentrated version of AEC, but does not result in more spots. The background is typically higher. TMB, a HRP substrate, seems to be the most sensitive substrate, resulting in somewhat higher spot counts.

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