Lumit® Anti-Tag Reagents for Protein Interactions Technical Manual
Instructions for Use of Product(s)
W1600, W1620, W1640, W1660
Literature # TM723
Lumit® Anti-Tag Reagents for Protein Interactions are homogeneous (no-wash) immunoassays to measure protein:protein or protein:small molecule interactions. These reagents function by combining immunodetection and NanoLuc® Binary Technology (NanoBiT). NanoBiT® luciferase is a structural complementation reporter ideal for interaction studies. The NanoBiT® system is composed of two subunits: Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 11 amino acid peptide) that can be expressed as recombinant protein fusions or chemically conjugated to antibodies or other target proteins of interest. The LgBiT and SmBiT subunits have been optimized for stability and minimal self-association due to their weak affinity (Kd = 190μM). When LgBiT and SmBiT are brought into proximity, they form a functional enzyme that generates luminescence in the presence of a Nano-Glo® substrate.
Lumit® Anti-Tag Reagents for Protein Interactions interact with glutathione-S-transferase (GST), FLAG® or polyhistidine (6-His) tags via tag-specific antibodies chemically conjugated to LgBiT or SmBiT. Similarly, biotinylated molecules can interact with LgBiT- or SmBiT-conjugated streptavidin. When two proteins or a protein and small molecule with different tags are in proximity, the interaction can be detected using the corresponding SmBiT- and LgBiT- conjugated antibodies or streptavidin. The Lumit® Anti-Tag Reagents provide a biochemical immunoassay format for simple, high-throughput screening of protein:protein or protein:small molecule interactions.
Summary of Changes
The following changes were made to the 11/24 revision of this document:
- Corrected section number references in Sections 4.B, 5.B and 5.C.
- Corrected Anti-Tag Reagent Master Mix concentration in Section 4.C and 5.B.
- Corrected figure references in Sections 5.A and 5.B.
- Updated Steps 1 and 2 under Preparing Samples and Step 2 under Performing the Lumit® Protein:Small Molecule Interaction Immunoassay in Section 5.C.
- Updated Figure 14.
Revised 11/24.