Protein Fragment Complementation Analysis Service
Protein Fragment Complementation Assay (PCA) is a powerful tool for studying protein-protein interactions. The fundamental principle of PCA involves splitting a functional protein into two fragments, each of which is fused with one of the target proteins under investigation. When these two target proteins interact within the cell, the fragments come into proximity and complement each other, restoring the functional activity of the original protein. By detecting the reconstituted protein activity, researchers can determine whether an interaction occurs between the target proteins. Currently, studies primarily employ fluorescent fusion proteins to enable intracellular signal transduction and the construction of biochemical protein interaction networks. This approach is known as Bimolecular Fluorescence Complementation (BiFC). MtoZ Biolabs offers a comprehensive protein fragment complementation analysis service including labeling, post-labeling enrichment, mass spectrometry detection, and data analysis, providing researchers with an end-to-end solution for studying protein-protein interactions.
Gnanasekaran, P. et al. Methods Mol Biol, 2023.
Figure 1. BiFC Schematic Diagram.
Note: When two target proteins interact within the cell, the two fluorescent protein fragments come into proximity and complement each other, restoring the functional activity of the original protein and emitting fluorescence.
Services at MtoZ Biolabs
Leveraging an advanced analytical platform, MtoZ Biolabs has developed multiple protein fragment complementation analysis service platforms to meet various research needs. Our primary techniques include the following:
1. Conventional Bimolecular Fluorescence Complementation (BiFC) Technology Based on Fluorescent Proteins
Using fluorescent protein tags such as Venus, GFP, YFP, and Citrine, target proteins are labeled for interaction analysis. When two target proteins interact, the fluorescent protein fragments come into close proximity and complement each other, forming a fully functional fluorescent protein. Upon excitation by light, this reconstituted protein emits a fluorescence signal, enabling the detection of protein-protein interactions.
2. Multicolor Fluorescence Complementation Technology
Based on multicolor fluorescence complementation technology, MtoZ Biolabs offers a protein fragment complementation analysis service that enables simultaneous detection of multiple protein interactions within a single cell. The experimental principle involves splitting multiple fluorescent proteins (such as GFP, CFP, BFP, etc.) at specific positions and fusing them with target proteins to form fusion proteins. Through interactions between different target proteins, distinct fluorescent protein fragments complement each other, forming multiple protein complexes. By observing fluorescence of different colors, researchers can determine the interactions among multiple protein groups.
3. BiFC-FRET
Each of the two target proteins is fused with either the N-terminal or C-terminal fragment of Venus fluorescent protein. When these target proteins interact, the Venus protein is reconstituted, restoring its fluorescence (BiFC system). A third target protein is fused with Cerulean to form a fusion protein. If this fusion protein interacts with the heterodimer formed by the two target proteins, Cerulean can act as a donor, transferring energy to the reconstituted Venus fluorescent protein via Förster Resonance Energy Transfer (FRET). This system enables the simultaneous detection of interactions among three proteins, providing insights into complex protein interaction networks.
Shyu, Y J. et al. Microscopy and Microanalysis, 2007.
Figure 2. BiFC-FRET Schematic Diagram.
Applications
1. Studying Protein-Protein Interactions
The protein fragment complementation analysis service is primarily used to study protein-protein interactions. To date, various BiFC systems have been successfully applied to investigate interactions between proteins in diverse biological systems, including in vitro assays, viruses, Escherichia coli, yeast cells, filamentous fungi, mammalian cells, plant cells, and even at the whole-organism level.
2. Drug Development
The protein fragment complementation analysis service can also be utilized in drug development. By selecting key disease-associated proteins, stable BiFC-expressing cell lines can be established. These cells are subjected to different drug treatments, and the fluorescence signal intensity is monitored to assess changes in protein interactions. This approach demonstrates promising potential for screening and identifying potential therapeutic drugs.
Deliverables
1. Comprehensive Experimental Details
2. Materials, Instruments, and Methods
3. Data Analysis, Preprocessing, and Estimation
4. Raw Data Files
MtoZ Biolabs, an integrated Chromatography and Mass Spectrometry (MS) Services Provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. Free project evaluation, welcome to learn more details!
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