I'm conducting protein-protein interaction studies using a LexA-based Bacterial-Two Hybrid system with E. coli reporter strains. We employ MacConkey agar and β-galactosidase assays to evaluate the interaction strength of our proteins. I've obtained some interesting results that I would like to understand better.
In the MacConkey agar assay, the interaction appears intermediate between our negative and positive controls, suggesting a moderate level of interaction. However, the β-galactosidase assay shows that our protein interaction pair represses reporter gene expression more than even our positive control, indicating a stronger interaction. This assay measures absorbance at 420 nm to calculate β-gal activity in Miller units, while the MacConkey agar results are qualitative, based on colony color comparison.
Here are a few key protocol details:
I am curious about what might cause these differences in results between the two assays. I wonder if it could it be related to:
1. The different growth phases (Mac. agar/18-24 hrs vs. LB broth-to OD 0.5)
2. The specific analytes measured (lactic acid production/pH indicator vs. OPG/yellow product- max abs. 420 nm)
......or something else?
I would greatly value any suggestions for specific literature or search terms that could guide my further investigation into these observations. As I'm still defining the aspects of this issue that are unclear to me, recommendations on relevant topics or key areas of research would also be incredibly helpful. What should I focus on to gain a better understanding of these results?
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