Research Area - Biochemistry

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FRET analysis of protein-protein interaction and redox sensor folding using the ImageStream (09-005)


Merck:/Freestyle/BI-Bioscience/Cell-Analysis/amnis/Amnis-Research-Images/biochemistry.jpgClassical biochemical techniques are often employed to measure location and co-location of molecules, tasks ideally suited for Amnis® imaging flow cytometry system. Examples include molecular translocation of transcription factors from the cytoplasm to the nucleus, trafficking of molecules to sub-cellular organelles, co-localization of proteins on, in, or between cells, just to name a few. The Amnis® imaging flow cytometry's ability to obtain statistically robust per-cell measurements of probe location and co-localization within highly heterogenous samples provides significant advances that complement and extend traditional biochemical research.

NF-kB Translocation in T:APC Conjugates

Measurement of NFκB translocation in transgenic T cells which are in contact with antigen presenting cells (APC) and specific peptide. Specific T cell:APC conjugates are identified and translocation of NFκB from the cytoplasm to the nucleus specifically within the T cells is measured in the presence (shown) or absence of peptide. See the application note for more details.

Co-Localization of an Antibody-Drug Conjugate to Endosomes or Lysosomes

In this experiment we use the capabilities of the Imagestream®x system to measure the transit of a fluorescently labeled antibody-drug conjugate (ADC) through the cellular endocytic pathway. The high spatial resolution afforded by the Imagestream®x system allowed accurate measurement of ADC co-localization to endosomes and lysosomes. See the application note for more details.

FRET Using the Imagestream®x for Detection of Protein-Protein Interactions

In fluorescence microscopy, light at one wavelength is absorbed by a fluorophore and emitted at a longer wavelength. FRET (Fluorescence or Forster Resonance Energy Transfer) can occur when a second fluorophore is in very close contact such that it can accept the energy from the first fluorophore and emit the light at an even longer wavelength. The efficiency of the transfer is extremely sensitive to the separation distance between the fluorophores and therefore when the emission is detected at the longer wavelength the conclusion is that the fluorophores were within approximately 10 nanometers of each other. When two fluorophores (a donor and acceptor pair) are used to label two different proteins, the close proximity of the two proteins are inferred by the measurement of the FRET from the donor to the acceptor fluorophore. The combination of high speed image acquisition and automated quantitative image analysis with FRET on the Imagestream®x allows measurement of the spatial location of the protein interaction within the cell or between cell conjugates even for rare subpopulations. Distinguishing intracelluar location of FRET vs. location of the proteins when not exhibiting FRET behavior offers a major advancement in understanding signaling pathways.

In this experiment receptor 1 is labeled with PE (donor) and receptor 2 is labeled with AF647 (acceptor). Cells were stimulated or not-stimulated and images collected with 488nm laser excitation. The graph shows that FRET to the AF647 fluorophore can be detected in the stimulated sample.