Nano-Glo® HiBiT Blotting System Technical Manual

Instructions for Use of Product(s)

Literature # TM524

The Nano-Glo® HiBiT Blotting System simply and sensitively detects HiBiT-tagged proteins on nitrocellulose or PVDF membranes following SDS-PAGE and transfer—no antibodies needed. HiBiT is an 11-amino-acid peptide tag that can be fused to the N or C terminus of the protein of interest or inserted into an accessible location within the protein structure. The amount of HiBiT-tagged protein transferred to the membrane is determined by adding a reagent containing the substrate furimazine and Large BiT (LgBiT), the large protein subunit used in NanoLuc® Binary Technology (NanoBiT®). HiBiT binds tightly to LgBiT (KD = 0.7nM), spontaneously forming a complex that generates a bright, luminescent enzyme. When added to a protein blot, LgBiT only generates luminescence when bound to immobilized HiBiT, virtually eliminating background caused by nonspecific binding. HiBiT-tagged proteins generate a proportional signal over five orders of magnitude, down to femtogram amounts. The result is a blotting system that yields sensitive detection of HiBiT-tagged proteins with greater speed and simplicity than protocols that use multiple steps for antibody blocking, binding and washing.

Printed in the USA 8/17.