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ELISA | Troubleshooting Forum

11/27/2012
Kristie Nybo, Ph.D.

Are there known compatibility issues with the hexa-histidine tag and certain ELISA plates?


BioTechniques Molecular Biology Forum brings bench scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the “Immunology and Immunochemistry Methods” forum.

Q: I am developing serodiagnostic assays for novel viruses by expressing proteins and using them to detect antibodies in human sera. The purity and yield of His-tagged proteins extracted under denaturing conditions has been good, but I see a lot of background and sporadic signals in ELISA using these proteins. Are there known compatibility issues with the hexa-histidine tag and certain ELISA plates?

A: The presence of His-tag in the antigen should not affect the ELISA signal if you block well. You might try nickel coated plates so that the His-tag…

A: High background and sporadic signals are caused by excess antibody, which increases nonspecific…

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