The Lab Notebook

Don't FRET- You've got Fluoppi!

Posted by Bindi M. Doshi, PhD on Sep 29, 2015 8:41:14 AM
Visualizing protein-protein interaction (PPI) is pretty neat. You are able to glimpse into a really unique aspect of nature that many people don’t get to see. It’s like watching lions and zebras interact but without the bloody violence.  However, anyone who has tried to visualize two proteins interacting can attest to how difficult it can be. It can involve long hours, uncertainty, and of course, photo bleaching. Blech! MBL’s new technology, Fluoppi, makes visualizing protein-protein interaction a little easier. This technology utilizes fluorescent tags that have a high signal:noise ratio. The only equipment you need is a microscope to observe fluorescence.
 
 
The first step to Fluoppi is to make fusions using two different tags:
1) Fuse protein X with FP tag to make X-FP
2) Fuse Protein Y with Ash to make Y-Ash
 
 
After making your fusion babies, you co-transfect your cells. The cytoplasm should “glow” or fluoresce. Then you add an inhibitor. The dispersed fluorescence in the cytoplasm re-distributes as fluorescent foci within the cell.
 
fluoppi_inhibition_induction.jpg
 
And who doesn’t like to have options? Here, you have 2 color options Azami green and Monti Red. CoralHue humanized Azami-Green (hAG) absorbs light at 492 nm and emits at 505 nm. Or try Monti Red! With this plasmid, light is absorbs at 571 nm and emitted at 607 nm, similar to Texas Red filters.
 
 
Using the Fluoppi system, the PPI interaction is also reversible. Remember, in nature, there are some proteins that interact with each other only transiently. They do not stay attached. Sometimes, they bind reversibly, assemble and disassemble, and rearrange as the cells needs for each specific function. So if you are trying to visualize proteins interacting with each other, you might want to know if they come together and then separate, or if they come together and stay together.
 
 
Compared to using FRET in living cells, Fluoppi is an easier to develop PPI detection system. FRET requires time consuming linker optimization. With Fluoppi, there are only 8 combinations of expression vectors to try. In addition, isolation of true positive FRET signal in living cells can be difficult, even for the experts.  However, by using Fluoppi, it is easy to find PPI by detecting fluorescent dot (foci) in cells.  The signal is either dispersed or it’s localized in foci. Easy peasy!
 
 
Fluoppi can be used for High-Throughput Screening (HTS) for screening different chemicals using high-content analyzers that might inhibit or induce an interaction between your two proteins of interest. Once you have your plasmids, you can easily seed many wells and gather lots of important data all at the same time.
 
 
Protein-protein interactions are so important in cell functions and regulations. Once we have a better understanding of these processes, we can develop better understanding of biological research that can lead to advancement in medical technologies.
 
View induction here:
 
 
 

 View inhibition here:

 

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Topics: Fluoppi, Fluorescent Proteins