Promega's Cookie Policy

We use cookies and similar technologies to make our website work, run analytics, improve our website, and show you personalized content and advertising. Some of these cookies are essential for our website to work. For others, we won’t set them unless you accept them. To find out more about cookies and how to manage cookies, read our Cookie Policy.

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.