Reporter based assay systems have turned out to be one of the most convenient ‘read outs’ for a variety of biological processes. A handful of reporter based assay systems are available in the market including luciferase, green fluorescent protein (GFP), red fluorescent protein (dsRed, tdTomato Red), beta-galactosidase etc. The reporter gene can be engineered with the protein of interest in such a way that it can be ‘constitutively active’ (always expressing) or can be induced to express under certain conditions of interest to the scientist. Luciferases are used as reporters in a variety of bio-applications such as neuroscience, immunology, oncology, virology as well as for monitoring biological processes in animal experiments.
The Gaussia Luciferase assay kit from New England Biolabs (NEB) is based on the properties of chemiluminescence for the signal readout. The Gaussia lucifearse has been developed from marine copepod Gaussia princeps. Other luciferases which are also commonly used as reporter genes include Firefly luciferase and Renilla lucifearse. The Gaussia lucifearse is much stronger in terms of the quantum yield of chemiluminescence (1000-fold higher) as compared to the Renilla or Firefly and offers the greatest advantage of being naturally secreted into the media and thus offering the ease of detection. The Gaussia lucifearse is the smallest lucifearse till date and is ATP-independent. The signal is detected by adding the substrate coelenterazine and measuring the emitted photons using a luminometer.
I have used this kit from NEB with outstanding success. My protein is not secreted in the media and thus I extracted the protein using a freeze-thaw protocol. Using the supernatant from the extracted protein and adding the substrate along with the assay buffer, my experiments have been successful. Instead of a luminometer, I have used a standard plate reader for the output signal and it works great (in my case, I have used the Synergy 2 plate reader from Biotek instruments). Qualitative signal detection can also be observed on a gel-documentation system using the chemiluminescence mode. Use of flat-bottom black or white color plates is recommended for best signal output and minimal loss. While taking the reading, it is best to do with minimum natural light interference from elsewhere (turn off the lights of the room, and make it as dark as possible). The reaction volumes suggested by the NEB kit works perfect for the signal detection. I have also engineered the Gaussia gene by splitting it into two and recombining the halves by expressing them together tagged to another protein (Bimolecular Chemilumnescence (BiLc) assay) in an assay and the signal is easily detectable under similar conditions too. I used the Gaussia plasmid from NEB itself for all the research.
The only thing which is a bit troublesome is addition of the substrate to one well at a time and measuring the output. This is recommended by the protocol, as the signal intensity is inversely proportional to the elapsed time. I have tried adding the reagent to multiple wells at the same time and taking the measurement, the difference noticed is not much significant if the whole process is done quickly and in an efficient manner.
Senior Scientist
Center for Neurodegeneration & Experimental Therapeutics (CNET) / Neurology
University of Alabama at Birmingham