Description
Measure Real-Time Abundance of Endogenously Regulated HiBiT-Tagged Proteins
Perform quantitative time-course studies on the same sample by adding a single reagent and simply measuring bioluminescence over time. No need to collect multiple samples, guess at which endpoints to measure, or develop antibodies against target proteins. There are several options for incorporating intracellular LgBiT into your CRISPR-Cas9 HiBiT knock-in workflow:
Use the HEK293 LgBiT Cell Line to create HiBiT-knock-in cell pools or clones. Additional LgBiT stable cell lines are also available through Promega custom assay services.
Create a stable LgBiT cell line of your choice using the LgBiT Expression Vector and further edit the cell line to knock-in HiBiT.
Edit HiBiT into the endogenous locus of your cell line of choice and create a stable cell line. Transfect the LgBiT Expression Vector to either create a stable LgBiT-expressing variant or transiently express the LgBiT protein.
Live-Cell, Kinetic Monitoring of Endogenous HiBiT-BRD4 Following PROTAC Treatment
HEK293 LgBiT Cell Line CRISPR-Cas9 Edited to Knock-In HiBiT
Transient LgBiT Expression Following HiBiT Knock-In into HEK293 Cells
Use Complemented HiBiT as the Energy Donor in NanoBRET® Applications for More Assay Possibilities
The intracellular LgBiT expression reagents allow HiBiT-tagged proteins to be used as the bioluminescent energy donor in live-cell NanoBRET® assays for the study of protein:protein interactions or small molecule target engagement. NanoBRET® technology is a proximity-based method dependent upon energy transfer from a bioluminescent energy donor protein to a fluorescent acceptor. Live-cell HiBiT complementation creates the bioluminescent donor, which can be coupled with different fluorescent acceptors to create live-cell interactions assays with HiBiT-tagged protein.
Ubiquitination Kinetics of Endogenously Expressed HiBiT-BRD4
HEK293 LgBiT Stable Cell Line was modified using CRISPR-Cas9 gene editing to create cells expressing a HiBiT-BRD4 fusion at the endogenous BRD4 locus. BRD4 ubiquitination levels were monitored following treatment with various PROTAC compounds by transiently transfecting the HaloTag®-Ubiquitin Fusion Vector. Energy transfer from the bioluminescent BRD4 donor to HaloTag®-Ubiquitin fluorescent acceptor was measured using the NanoBRET® Nano-Glo® Kinetic Detection System.
LgBiT Expression Vector Information
LgBiT Expression Vector sequence text file .
