Description
Detect LgBiT Subunit of NanoBiT® Complementation System
Anti-LgBiT Monoclonal Antibody is a protein A/G affinity-purified mouse monoclonal antibody that is used to detect Large BiT (LgBiT) and LgBiT fusion proteins via Western blotting and immunofluorescence. Weak cross-reactivity with NanoLuc® luciferase is observed.
LgBiT is the large 18kDa subunit of NanoBiT® complementation technology. LgBiT interacts with the low-affinity SmBiT or the high-affinity HiBiT peptides to form a functional luciferase. LgBiT protein fusions are used with SmBiT protein fusions for live cell analysis of protein interactions. Unfused LgBiT can also be expressed in cells to detect HiBiT-tagged proteins without the need for cell lysis.
Western Blot Results The figure on the right shows an example Western blot using HEK293 cells transfected with CMV-based expression constructs encoding LgBiT fusion proteins. The expected mobility was seen for each of the LgBiT fusion proteins. We recommend a concentration of 1µg/ml as a starting point for protocol optimization. Low levels of the LgBiT-KRAS fusion were detected even with CMV-driven expression and without dilution using Transfection Carrier DNA.
Detection of NanoLuc® fusion proteins expressed in HEK293 cells.
Immunofluorescence Results Shown below are example immunofluorescent images of HeLa cells transfected with a CMV-based construct expressing LgBiT-CRAF fusion protein. The LgBiT-CRAF fusion protein is stained red, and nuclei are marked blue via Hoechst staining. We recommend a concentration of 1-3µg/ml for immunofluorescence optimization. No LgBiT staining is observed in HeLa cells transfected with only carrier DNA (mock).
LgBiT-CRAF (3µg/ml Anti-LgBiT)
LgBiT
Hoescht
Merge
Mock (3µg/ml Anti-LgBiT)
