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Weak interactions in Y2H assay? Very sceptical

Y2H Protein biochemistry protein methods

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#1 FreddieP

FreddieP

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Posted 24 July 2013 - 05:29 AM

Hi, Thank you for reading my question.

These proteins interact positively in HIS- media but do not turn blue in the X-gal assay. According to the manufacturers instructions this indicates a positive but weak interaction. Other proteins interact positively in both the HIS and x-gal assay. The bait protein has a protein-protein interaction domain that is mostly proline so I am concerned about reporting a spurious interaction because of a promiscuous protein binding domain on the bait. The weak interactions were confirmed in a pull-down assay but this could still be artefactual. How can I be more sure of this weak interaction? Let me know as I am not very experienced in experimental protein:protein interactions. Thanks




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