Many, many techniques are available to assess protein-protein interactions. One popular approach is to fuse a protein of interest to each part of a split fluorescent protein (FP) and measure the signal produced when the candidate proteins’ interaction brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence Complementation (BiFC). It can provide a qualitative or quantitative readout of the interaction in vivo or in vitro, and can be used for measuring protein expression or localization in cells, or even single-particle tracking of the bound complex.
There are so many FPs available, with many ways to split and mix and match them, so where should you begin? Have no fear, Addgene is here to help!
General Design
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| Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein A and B interact, the FP fragments can assemble the full structure of the fluorescent protein, producing fluorescent signal. Image modified from Feng et al. 2017 under CC-BY license. |
Many split-FP fragments are named according to the number of strands in the canonical beta-barrel structure. Most frequently, the proteins are split between strand 10 and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible with multiple FP(11) fragments, even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked below only provide one fragment of the FP because they are used with an already-available complementary fragment.
Next, let's look at a round-up of some new and popular split-FP plasmids from our catalog to help you find the right split-FP plasmid for your experiment.
Highlighted Split-Fluorescent Protein Tags
Browse the table below, and click the Article link to find all the plasmids from the associated paper:
| Color |
Base FP |
Description |
Article |
PI |
| Blue |
EBFP2 |
EBFP2(1-10) and Capri(1-10) for use with GFP(11) |
Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R, Jiang F, Xie J, Kamiyama D. Commun Biol. 2021 |
Daichi Kamiyama |
| Cerulean |
Cerulean(1-10) for use with GFP(11) |
Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R, Jiang F, Xie J, Kamiyama D. Commun Biol. 2021 |
Daichi Kamiyama |
| ECFP |
C155 of ECFP pairs with N173 derived from Cerulean or Venus |
Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 |
Chang-Deng Hu |
| Green |
sfGFP |
Split super-folder GFP, our most-requested GFP(1-10) and GFP(11) |
Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. Nat Commun. 2016 |
Bo Huang |
| spGFP |
Split superpositive GFP |
Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo. Blakeley BD, Chapman AM, McNaughton BR. Mol Biosyst. 2012 |
Brian McNaughton |
| mNeonGreen2 |
mNG2(1-10) and mNG2(11) |
Improved split fluorescent proteins for endogenous protein labeling. Feng et al. Nat Commun. 2017 |
Bo Huang |
| mNeonGreen3 |
mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) |
Improved yellow-green split fluorescent proteins for protein labeling and signal amplification. Zhou S, Feng S, Brown D, Huang B. PLoS One. 2020 |
Bo Huang |
| Yellow |
Venus |
pBiFC-VN173, pBiFC-VC155 |
Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques. 2006 |
Chang-Deng Hu |
| Venus |
Improved N-terminal fragment VN155(I152L), for use with VC155 |
An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Kodama Y, Hu CD. Biotechniques. 2010 |
Chang-Deng Hu |
| mVenus |
pET-BiFC contains both fragments of split mVenus (aa 155), includes I152L in N-terminal fragment |
An enhanced recombinant amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting alpha-synuclein oligomerisation. Eastwood T, Baker K, Brooker H, Frank S, Mulvihill DP. FEBS Lett. 2017 |
Dan Mulvihill |
| mVenus |
Split mVenus (aa 155), includes I152L in N-terminal fragment |
Constant rate of p53 tetramerization in response to DNA damage controls the p53 response. Gaglia G, Lahav G. Mol Syst Biol. 2014 |
Galit Lahav |
| EYFP |
Gateway vectors with EYFP split at 175 |
Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Lu et al. Plant J. 2010 |
Yuhai Cui |
| Red |
mScarlet |
Split-wrmScarlet variant of mScarlet |
Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans. Goudeau et al. Genetics. 2021 |
Cynthia Kenyon |
| mRuby4 |
mRuby4(1-10), pairs with sfCherry2(11) |
Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R, Jiang F, Xie J, Kamiyama D. Commun Biol. 2021 |
Daichi Kamiyama |
| sfCherry2 |
sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) |
Improved split fluorescent proteins for endogenous protein labeling. Feng et al. Nat Commun. 2017 |
Bo Huang |
| sfCherry3 |
Improved sfCherry3C(1-10) for use with sfCherry2(11) |
Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses. Feng S et al. Commun Biol. 2019 |
Bo Huang |
| Near-Infrared |
iRFP |
iSplit, comprised of PAS and GAF domains of iRFP713. Requires presence of heme oxygenase for chromophore formation (abundant in eukaryotes) |
A Near-Infrared BiFC Reporter for In Vivo Imaging of Protein-Protein Interactions. Filonov GS, Verkhusha VV. Chem Biol. 2013 |
Vladislav Verkhusha |
| iRFP |
GAF domains from miRFP709 and miRFP670, pair with common PAS domain. Requires presence of heme oxygenase for chromophore formation (abundant in eukaryotes) |
Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Shcherbakova et al. Nat Commun. 2016 |
Vladislav Verkhusha |
| Multiple |
FAST |
Fluorescence-activating and absorption shifting tag (FAST) for use with green-yellow or orange-red fluorogen |
A split fluorescent reporter with rapid and reversible complementation. Tebo AG, Gautier A. Nat Commun. 2019 |
Arnaud Gautier |
| HaloTag |
TagBiFC allows exogenous labeling for single-molecule imaging |
TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells. Shao S, Zhang H, Zeng Y, Li Y, Sun C, Sun Y. Commun Biol. 2021 |
Yujie Sun |
For even more options, check out the 2in1 Kit for FRET and ratiometric BiFC from Christopher Grefen’s lab or explore the MoBiFC modular bimolecular fluorescence complementation toolkit from Benjamin Field’s lab.
Special Considerations
There are a few special exceptions to the usual split-FP options. While the BiFC interaction is typically irreversible and requires some time for chromophore maturation, splitFAST displays rapid and reversible complementation. TagBiFC (split HaloTag) provides maximum flexibility in color labeling with exogenous fluorophores, which can even be bright enough for single-molecule tracking in live cells.
Controls
When designing experiments, it’s important to include proper controls to assess the behavior of the split FP in your system of interest. Remember that the FP fragments themselves can sometimes interact in cells and produce background signal (especially for some older split-GFP constructs or certain cell environments). Also consider that a protein fusion may disrupt functionality or binding of your target proteins, especially if the termini are important for their interaction. You can read our previous blog post for more on BiFC principles and experimental design.
We hope you find these plasmids useful. Good luck, and happy experimenting!
References and Resources
Additional resources on the Addgene blog
Resources on Addgene.org
Reference
Feng, S., Sekine, S., Pessino, V. et al. Improved split fluorescent proteins for endogenous protein labeling. Nat Commun 8, 370 (2017). https://doi.org/10.1038/s41467-017-00494-8. PMID: 28851864.
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