02 August 2017
- Last edited 02 August 2017
Thanks for your question and sorry for delay in getting back to you.
In general, known interaction pairs are 10-1,000 fold higher than the LgBiT fusion co-expressed with HaloTag-SmBiT. This is only a guideline, however, and we’ve seen cases where the signal is actually dimmer. How much brighter are the different orientations vs. a mock-transfected control? If you’re not well above this control, you could consider a switch to CMV-based constructs. For the PKA pair, is the LgBiT-PRKAR2A:SmBiT-PRKACA pair 50-60 fold brighter than the LgBiT-PRKAR2A:HaloTag-SmBiT control?
For a switch to CMV-based constructs, I’d recommend our new BiBiT-ready vectors, which we have for both MCS and Flexi formats. Once you identify an optimal pair, you can use HygR and BlastR resistance genes on separate plasmids to make a stable cell line. Alternatively, you can digest both plasmids and ligate fragments from each to make a single construct where both fusions are expressed via a bi-directional CMV promoter. This is the “BiBiT” vector, which has the BlastR resistance gene, and this is our preferred approach for stable expression. The BiBiT-ready vectors are currently available via our Custom Assay Services department (Promega CAS). Because they use the CMV promoter, it will be important to dilute the plasmid DNA when using transient expression as described in Section 5.B of TM461. Without dilution, you’ll very likely see low S:B for treatments that are known/expected to modulate the interaction. If the BiBiT system isn't of interest you could also move your constructs into any CMV vector that you currently have.
I hope this helps but please let us know if you have additional questions.