| Assay Method Information | |
| | Fluorescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of RecA-Intein Splicing Activity |
| Description: | Keywords: GFP-RecA intein, protein splicing, refolding, reducing reagent, GFP fluorescence Assay Overview: M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. For primary screening, RecA intein fusion protein was incubated with compounds first in 1536 well plates. The RecA intein splicing activity was initiated by addition of reducing reagent TCEP. GFP fluorescence, produced as a result of protein splicing, was read with fluorescence plate reader. Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to inhibition of RecA-Intein splicing activity. |
| Affinity data for this assay | |
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