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NanoBiT® Protein:Protein Interaction System Technical Manual

Instructions for Use of Product(s)
N2014, N2015

Literature # TM461

NanoLuc® Binary Technology (NanoBiT™) is a two-subunit system based on NanoLuc® luciferase that can be used for intracellular detection of protein:protein interactions (PPIs). Large BiT (LgBiT; 17.6kDa) and Small BiT (SmBiT; 11 amino acids) subunits are fused to proteins of interest, and when expressed, the PPI brings the subunits into close proximity to form a functional enzyme that generates a bright, luminescent signal. Unlike related approaches, where an enzyme or protein is divided into two fragments, LgBiT was independently optimized for structural stability and SmBiT was selected from a peptide library specifically for the PPI application. The result is a subunit pair that weakly associates (KD = 190μM) yet retains the bright signal of NanoLuc® luciferase at saturation. The interaction of LgBiT and SmBiT is reversible, and the system can be used to detect rapidly dissociating proteins. PPI dynamics can be followed in real time inside living cells using the Nano-Glo® Live Cell Assay System, a nonlytic detection reagent that contains the cell-permeable substrate furimazine.

Summary of Changes
1. Corrected reference to Step 1 in Section 6.C, Option #1, Step 5 and Option #2, Step 3.
2. Updated NanoBiT® to registered trademark information.
3. Corrected FuGENE® HD Database URL.

Printed in USA. Revised 9/16.