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3CLglow Collection

Live cell assays for SARS-CoV-2 3CL protease

Montana Molecular has developed three new SARS-CoV-2 3CLpro biosensor assays in response to the COVID-19 pandemic. The assays provide robust, biologically meaningful tools to evaluate therapeutic drugs that target SARS-CoV-2 replication.

All our sensors are delivered with a single BacMam vector, and the kits themselves are BSL-1 and easy to use.


  • 3CLglowUp: Our brightest 3CLpro assay – this is a robust 3CLpro inhibitor assay that is easily detected on plate readers. Green fluorescence increases when 3CLpro is successfully inhibited.
  • 3CLglow: The original iteration of our 3CLpro assay – this tool expresses both a green fluorescent sensor for 3CLpro activity and a constitutively expressed red fluorescent protein to control for cell health and compound toxicity. Green fluorescence decreases with 3CLpro inhibition. It is also compatible with plate readers.
  • 3CLglowLive: This a SARS-CoV-2 3CLpro live virus assay. With extremely low background fluorescence, the 3CLglowLive reporter increases fluorescence in the presence of viral replication, or 3CLpro. It is best suited for microscopy experiments where the experimenter needs to be able to rapidly identify if there is viral replication without taking a fluorescent measurement.

3CLglowUp detecting nirmatrelvir inhibition against mutated 3CL proteases

A figure comparing IC50 of nirmatrelvir vs. C5a in Caco-2 cells using a fluorescent 3CL pro biosensor.

3CLglow expressed in HEK293 cells, showing green and red fluorescence in response to nirmatrelvir and C5a.

3CLglowLive expressed in HEK293 cells, with and without 3CLpro co-expression.

Simple 3CLpro Inhibitor Assay Steps

  • Day 1 – Plate cells.
  • Day 2 – Make transduction mix by combining 3CLpro sensor BacMam, 3CLpro Enzyme, complete media, and sodium butyrate. (3CLpro sensor BacMam, 3CLpro enzyme BacMam, and sodium butyrate are included in our 3CLglow sensor kits)
  • Day 3 – Add inhibitor compounds and measure fluorescence in either an endpoint assay to maximize throughput, or a kinetic assay for real-time detection of inhibitor action in living cells.

Recommended Kits for inhibitor assays: 3CLglow (C1160G) or 3CLglowUp (C1161G)

Figure showing the general mechanism of the 3CLglowUp assay. The sensor is dim in the presence of 3CLpro, but increases in fluorescence with successful inhibition.

Measure inhibition of mutant 3CL proteases

Dose-response curves for nirmatrelvir inhibition of 3CLpro and mutant proteases C1206N-C1215N

  • Mutations to the 3CLpro enzyme have been found to decrease the efficacy of existing therapeutics like Nirmatrelvir. Montana Molecular has packaged a suite of mutant proteases in BacMam for easy co-expression with our 3CLpro biosensors.
  • Screen for the next generation of 3CL protease inhibitors that are effective against the mutated SARS-CoV-2 virus.
  • Compatible with detection on fluorescence plate readers or imaging systems.

3CLglowLive – Detect viral replication

    • Test the effectiveness of antibodies to block viral entry
    • 3CLglowLive has extremely low background fluorescence, allowing rapid identification of cells undergoing viral replication (3CLpro activity)
    • Compatible with fluorescence imaging systems.

    Cells transduced with 3CLglowLive and infected with SARS-CoV-2 virus, varying PFU.