Updated February 15, 2021
If you’re interrogating two proteins to understand the conditions under which they interact, a complementation system enables you to tag each protein. Interaction of the tagged proteins facilitates the complementation of the subunits, resulting in a signal. Here we discuss the NanoBiT complementation assay and describe its use to study mitochondrial fission.
About the NanoBiT™ Complementation Assay
However, not all split firefly luciferase systems are created equal.
NanoBiT™ Complementation Reporter is a protein interaction assay that features the improved NanoLuc® luciferase. NanoLuc luciferase, originally isolated from a deep sea shrimp, is a small luciferase that provides a much brighter signal than firefly luciferase.
Using NanoLuc luciferase, two NanoBiT subunits have been developed and independently optimized for stability and minimal self-affinity, as well as small size (18kDa for LgBiT and 11 amino acids for SmBiT). The benefits of NanoBiT include:
• small subunits that are not likely to interfere with protein interactions
• a bright signal that results in a greater signal:background ratio and thus a more sensitive assay.
Applying the NanoBiT Complementation Assay to Mitochondrial Fission
Dr. Stefan Strack, an investigator at the University of Iowa Carver College of Medicine, discusses his studies on mitochondrial fission using a split luciferase in this chalk talk. The goal of his work is to learn more about the mechanism of mitochondrial fission.
Mitochondria and Disease
Mitochondria are organelles found in abundance in most cells of plants and animals. Often called the cells’ powerhouse, mitochondria are where cellular respiration takes place; energy in the form of ATP is created when mitochondria intake and metabolize breakdown products of carbohydrates and fatty acids.
Not surprising given their importance in energy metabolism, dysfunction of mitochondria has been associated with serious diseases such as muscular dystrophy, Parkinson’s, Huntington’s and Alzheimer’s disease, as well as conditions that can result in blindness.
Strack’s group is interested in interactions of mitochondrial outer membrane proteins Drp-1 and mff (mitochondria fission factor).
Why Use NanoBiT Complementation?
The Strack laboratory decided to try the new NanoBiT™ complementation assay, and realized that the better NanoBiT signal to background ratio compared to the split firefly luciferase, gave them a much more sensitive assay.
In addition, the small size of the NanoBiT subunits means that the Strack team doesn’t have to worry about the two subunit tags interfering with normal mitochondrial protein function.
You can learn more about the NanoBiT Complementation Assay, see other videos, view data, and even join a forum to discuss your questions and results with others, including product experts, here.
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