Bimolecular Fluorescence Complementation (BiFC)

Bimolecular Fluorescence Complementation (BiFC) is a technology developed based on protein complementation that can be used to detect protein interactions in vivo or in vitro. The principle is that fluorescent proteins can be separated from specific sites to produce two non-fluorescent active fragments, N-fragment and C-fragment. When the two fragments are fused to the interacting proteins (protein A and protein B), they will be pulled closer due to the interaction force of the proteins, and complementary to each other, to reconstruct into an active fluorescent protein and generate fluorescence under excitation light. Therefore, through the visualization and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest. ^[1]

Fig.1 Schematic diagram of bimolecular fluorescence complementation principle.

• Expression vector construction
  Choose an appropriate fusion protein production system, determine fusion sites, and design linkers. Then create a suitable plasmid expression vector.
• Cell culture
  Culture cells in an appropriate medium for vigorous growth following transfection or transduction. Adherent cells are most easily visualized by microscopy, while non-adherent cells can be analyzed by flow cytometry.
• Cell transfection
  Cells were transfected with appropriate amounts of plasmids or transduced with viruses encoding putative interacting partners fused to complementary fluorescent protein fragments such as A-YN155 and B-YC155. A minimal amount of plasmid or virus is used to generate a detectable signal.
• Imaging of cells
  The cells are cultured so that the protein is expressed. Visualize complexes as soon as possible after transfection to reduce the possibility that fusion protein expression affects cell identity. The BiFC complexes were visualized using a fluorescence microscope with an appropriate objective. Confirm that cells show normal and/or expected morphology.
• Fluorescence quantitation
  Using fluorescence microscopy or flow cytometry, quantify BiFC complex formation by measuring the fluorescence intensity of BiFC complexes and intact fluorescent proteins in the same cells.
• Analysis of data
  The efficiencies of fluorescence complementation are determined by the value obtained by division of the intensities of fluorescence complementation and the intensities of whole fluorescent protein in each cell.

• Flexible: it can be used for both in vivo interaction studies and in vitro interaction studies.
• Direct visualization: the interaction results can be directly observed under the microscope.
• Wide applicability: the BiFC system has been successfully applied to different host cells such as animals, plants, fungi, and bacteria.
• Sensitivity: the verification result only needs to detect the presence or absence of fluorescence. The background is clean, and the sensitivity is high.
• No specialised equipment: it has low requirements for instruments, controllable cost, and relatively simple data processing.

• Proteins: study of protein interactions ^[2], study of the localization of protein interactions, and study of protein configuration.
• Drugs: screen of potentially therapeutic drugs.
• RNA: specific marker of RNA in cells.

Thermal Cyclers

Thermal Cycler is a laboratory apparatus that amplify segments of DNA using PCR. A thermal cycler is also commonly called a DNA Amplifier, PCR Machine, or Thermocycler. The cycle normally lowers or raises the block’s temperature in preprogrammed discrete steps. A thermal cycler is important for a laboratory dealing in molecular biology and gene cloning.

PCR Workstation / PCR Hood

A PCR workstation, also called a PCR hood, is a work space, enclosed on three sides, that provides a space for doing amplification of DNA and/or RNA. PCR hoods are used in biology and genetic labs so that there isn’t any cross contamination between samples. PCR workstations have no circulation, which helps to prevent contamination, and UV lights for sterilization.

PCR Consumables

PCR consumables generally involve pipettes, 100ul, 500ul and 1ml centrifuge tubes, 12 consecutive rows of tubes, and tube racks (96-well plates, etc.) for placing the centrifuge tubes. It is best to perform PCR experiments on ice, so you need to prepare an ice box. We use a wide variety of PCR consumables for you to choose from to meet your different needs.

Cell Culture Systems

With the ability to reduce human error and manhours, automated systems are crucial. However, large-scale coordination of the isolation and growth of cells in a controlled, sterile environment with temperature and humidity control can be daunting. To combat this, Cell Culture systems are designed to meet your individual needs and reach your automation goals.

Laboratory Incubators

An incubator is a device used to grow and micro biological cultures or cell cultures. It is made up of a chamber with a regulated temperature. Some incubators also regulate humidity, gas composition, or ventilation within that chamber. An incubator maintains optimal temperature, humidity, and other conditions such as the oxygen content of the atmosphere inside.

Cell Culture Dishes

Cell culture dishes are commonly used as experimental consumables for culturing microorganisms or cell cultures. They consist of a flat disc-shaped bottom and a cover. Designed specifically to aid in cell culture growth and propagation, cell culture dishes are offered in a myriad of formats, materials, and surface modifications.

Flow Cytometry

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. It is most commonly used to evaluate bone marrow, peripheral blood and other fluids in your body.

Electroporation

Electroporation refers to the use of short high voltage pulses to overcome cell membrane barriers. Transient and reversible breakdown of the membrane can be induced by applying an external electric field, which just surpasses the capacitance of the cell membrane. Electroporation is now used to deliver a large variety of molecules: from ions to drugs, dyes, tracers, antibodies, oligonucleotides to RNA and DNA.

Fluorescence Microscopes

A fluorescence microscope is an optical microscope that uses fluorescence instead of other light properties (such as scattering, reflection, and absorption) to generate an image. Here you will find superior pricing and a great selection of quality instruments for your research laboratory. We have the knowledge and expertise to assist you in making an optimal selection.

STEMart provides you with a variety of bimolecular fluorescence complementation equipment to meet your various R&D and application needs. If you have any questions or requirements for bimolecular fluorescence complementation equipment, please feel free to contact us.

References

• Rose RH, Briddon SJ, Holliday ND. Bimolecular fluorescence complementation: lighting up seven transmembrane domain receptor signalling networks. Br J Pharmacol. 2010 Feb; 159(4): 738-50. Doi: 10.1111/j.1476-5381.2009.00480.x. Epub 2009 Dec 10. PMID: 20015298; PMCID: PMC2829200.
• Miller KE, Kim Y, Huh WK, Park HO. Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies. J Mol Biol. 2015 Jun 5; 427(11):2039-2055. Doi: 10.1016/j.jmb.2015.03.005. Epub 2015 Mar 12. PMID: 25772494; PMCID: PMC4417415.