Visualize KRAS Protein-Protein Interactions on the GloMax® Galaxy Bioluminescence Imager

If you're used to working with bioluminescence, you’ve probably spent a fair amount of time explaining to others how it works, how it has superior sensitivity to traditional fluorescence assays, and why it’s your go-to method for detecting complex biological processes. But let’s be honest: no matter how impressive the data, there’s always been that lingering question—If the bioluminescent signal is generated through the summation of signal from a well of cells, how accurately does it reflect what's happening in a cell-to-cell basis?

That’s the kind of challenge often faced in the lab, where researchers rely on population-level data that, while valuable, is ultimately an approximation of what’s happening at the cellular level. Enter the GloMax^® Galaxy Bioluminescence Imager—a microscope that moves beyond simple numerical reports to offer something needed in the bioluminescent research space: the ability to see protein-protein interactions in real time, at the individual cell level.

Fig 1. GloMax^® Galaxy Bioluminescence Imager.

Bioluminescent reporters were traditionally used to monitor gene expression. NanoLuc^® has expanded the capabilities far beyond that. By enabling the creation of protein of interest (POI)-reporter gene fusions, NanoLuc^® Technologies provides researchers with the means to track protein dynamics—such as localization, degradation, and target engagement—within live cells. However, up until now, visualizing these intricate protein behaviors within a cell population has been a significant hurdle.

With the GloMax^® Galaxy, you can finally visualize bioluminescent events as they occur, offering unprecedented insight into cellular dynamics that were once obscured by the limitations of traditional microscopy. In this article, we dive into the real-world application of NanoBRET^® technology, using KRAS-CRAF interactions as a model to demonstrate how bioluminescence imaging is opening new doors for understanding protein behavior in live cells.

 

NanoBRET^® (Bioluminescence Resonance Energy Transfer) is an advanced method for detecting protein-protein interactions in live cells. It relies on an energy transfer between a NanoLuc® donor enzyme, and a HaloTag^® fluorescent acceptor protein. As the two proteins get into proximity, energy is transferred from the bioluminescent donor to the fluorescent acceptor. This energy transfer can be detected by measuring the light emitted by the acceptor, providing a real-time readout of protein interaction dynamics. This occurs without the need for external light stimulation, and thus results in much less background noise compared to traditional fluorescent assays.

Fig 2. Schematic on how the NanoBRET® Technology enables the observation of protein-protein interactions.

In our experiment, we employed NanoBRET^® to visualize the interaction between KRAS(G12D) and CRAF in living cells. KRAS is involved in regulating cell division, differentiation, and apoptosis through the RAS pathway¹. Mutations in KRAS, particularly the G12D mutation, are common in cancers and result in constitutively active KRAS, which drives uncontrolled cell growth. KRAS interacts with CRAF, a downstream effector in the RAS-RAF-MEK-ERK pathway, making this a biologically relevant system to study protein-protein interactions.

Fig 3. RAS-RAF-MEK-ERK pathway

To better understand CRAF, two domains were selected. The RBD (Ras Binding Domain) and CRD (Cysteine Rich Domain) of CRAF were chosen because of their high basal BRET signal when interacting with KRAS. HeLa cells were bulk transfected with NanoLuc-KRAS(G12D) and CRAF-RBD-CRD-HaloTag expressed from a bidirectional promoter (BiBRET), providing consistent long-term interactions between the two proteins.

The transfected cells were plated at a density of 30,000-40,000 cells per well in Ibidi microchambers and incubated overnight to allow for proper attachment and expression. These microchambers were chosen because their increased well depth is ideal for long-term kinetic imaging, providing better conditions for capturing detailed data over time. To visualize the interaction, the cells were labeled with Janelia Fluor^® HaloTag^® 549 ligand, which allowed us to track fluorescence, while NanoBRET^® Nano-Glo® Substrate was added to initiate the bioluminescent signal. Janelia Fluor® HaloTag^® dyes were selected due to them producing less background than traditional HaloTag^® dyes, which can interfere with accurate imaging.

Fig 4. NanoBRET^® Protein-Protein Interactions of NanoLuc-KRAS (G12D) Imaged on the GloMax^® Galaxy Bioluminescence Imager. The donor image shows bioluminescence captured after 180 second exposures. The acceptor image shows fluorescence capture of Janelia Fluor^® HaloTag^® Ligand 549. The rows indicate treatment with either 1x or 2x NanoBRET^® Nano-Glo^® Substrate.

Figure 4 displays filtered images of both the donor (bioluminescent) and acceptor (fluorescent) channels, captured after 180-second exposures using the GloMax^® Galaxy Bioluminescence Imager. A key factor in imaging NanoBRET^® interactions is optimizing the substrate concentration to generate the brightest and most stable signal. We tested a range of substrate doses, starting with the standard 1x dose, which produced a weaker signal. By increasing the substrate to a 2x concentration, we observed a significant increase in both the brightness and stability of the luminescent signal, ensuring more reliable data could be captured during the imaging process.

To further validate the use of NanoBRET^® for visualizing protein-protein interactions, we investigated the impact of MRTX-1133, a selective KRAS(G12D) inhibitor, on the KRAS-CRAF interaction. MRTX-1133 is a potent inhibitor known for its specificity to the KRAS(G12D) mutation, without affecting the expression of the KRAS protein itself². To initially quantify the effect of MRTX-1133 on KRAS activity, we generated a dose-response curve on the GloMax Discover (Figure 5). A BRET ratio was calculated by taking the acceptor signal and dividing it by the donor signal. A clear reduction in the BRET ratio was observed over a three-hour period, with an IC50 of approximately 1.4nM, confirming the potency of the inhibitor.

Fig 5. MRTX-1133 Treatment reduces NanoBRET^® Specific KRAS Activity

To determine what this treatment looked like on a cell-to-cell basis, we then captured images of cells treated with MRTX-1133 (Fig 6). To evaluate any bleedthrough of the donor signal into the acceptor channel, cells were tested without the HaloTag^® ligand, as shown in column 3. Control cells treated with DMSO produced bright and consistent bioluminescence and fluorescence signals, with a BRET ratio indicating strong energy transfer between KRAS and CRAF. Upon treatment with 1µM MRTX-1133 for four hours, we observed a significant reduction in the fluorescence signal (acceptor channel) while the bioluminescent donor signal remained unaffected. This shift in the BRET ratio—leaning heavily towards the donor signal— is shown in the bottom row with a much darker image of cells. This reduction in the BRET ratio suggests that KRAS expression persisted, but the protein-protein interaction with CRAF had been disrupted. 

Fig 6: Treatment With MRTX-1133 Blocks NanoBRET^® Acceptor Fluorescence. Donor row shows bioluminescence images. Acceptor row shows fluorescence images. Third row shows ratio between donor and acceptor responses within each cell. Strong BRET Ratio is indicated through lighter colors. No ligand control indicates the bleedthrough of donor signal into the acceptor channel.

 

One of the key advantages of using the GloMax^® Galaxy is the ability to distinguish between cells based on their individual responses. In this experiment, the imaging system allowed us to analyze the ratio of energy transfer at the single-cell level, providing deeper insights into how each cell responded to MRTX-1133 treatment. For example, some cells do show acceptor signal, suggesting they did not respond to the dose of MRTX-1133 selected. This highlights how subtle differences in cell responses can be missed when taking a snapshot of a global cell population.  

This capability offers significant advantages over traditional plate-based assays, which average out signals across a whole cell population. By detecting the signal at the level of individual cells, researchers can discern subtle variations in responses, enabling more detailed analysis of complex biological systems. The ability to measure these interactions visually brings a new dimension to data analysis—moving beyond numerical outputs to actual visualization of protein dynamics.

NanoBRET® technology provides a sensitive method for detecting protein-protein interactions but has historically been difficult to visualize due to limitations in bioluminescent imaging. The GloMax® Galaxy Bioluminescence Imager opens new possibilities to understand complex protein activity through real-time imaging of bioluminescent signals. 

The combination of NanoLuc® Technology and the GloMax® Galaxy offers unparalleled sensitivity, spatial resolution, and insight into cellular processes providing a way to tease apart the numbers generated on a plate reader. This expands the possibilities to view protein-protein interactions as they occur in real-time, improving workflows and increasing confidence in a numerical report by providing visualization of actual cellular events.

What does this mean for the lingering question? The answer is clear. Tools like the GloMax® Galaxy will play an essential role in bridging the gap between quantitative data and visual insights, continuing to drive disease and therapeutic discovery.

To learn about the GloMax® Galaxy Bioluminescence Imager, visit the Product Page.