Evaluation of Chemical Fluorescent Dyes as a Protein Conjugation Partner for Live Cell Imaging
Research Overview
In the area of cell biology, living cell fluorescence imaging is a powerful and commonly used technology. To obtain high resolution imaging results without damaging the cells, appropriate objective lenses, filter sets, and microscope detectors are adopted to optimize the imaging conditions. Apart from that, it is also of pivotal significance to choose the right fluorophore. In protein studies, small chemical fluorescent dyes play a part in tracing specific proteins, especially when conjugated with genetically encoded tags, such as HaloTag, SNAP, CLIP, and tetracystein. The effects of conjugation with fluorescent dye on protein function should be evaluated even though it is theoretically workable that any fluorescent dye can be used for living cell imaging. A recent study also revealed that the properties of dyes can affect the binding of the conjugated antibody.
Yoko Hayashi-Takanaka and other team members conducted a systematic evaluation of the suitability of various dyes on the market with different excitation wavelengths or intracellular fluorescence live imaging.
Research Procedures and Materials
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Antibodies and Fab fragment preparation
The mice were immunized with a synthetic peptide KQLATKAAR(me3-K)(phospho-S)APATGGVKC coupled to keyhole limpet hemocyanin, so as to produce a mAb targeting phosphorylated histone H3 Ser28 (H3S28ph. Fab fragments were prepared using a preparation kit, of which the binding affinity to the epitope was measured by surface plasmon resonance.
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Cell imaging and immunofluorescence
The team plated the cells on a glass-bottom dish and loaded them with fluorescent Fab fragments using glass beads (1 mg/ml Fab; 2 ml) or by microinjection, using Femtotip (0.2 mg/ml). And obtained fluorescent images by an inverted microscope with a PlanApo VC 1006 (NA = 1.4) oil-immersion objective lens under the operation of NIS Elements ver. 3.0.
The team collected the immunofluorescence images with a fluorescence microscope.
Results
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The effect of fluorescence:protein ratio on epitope binding of H3S28ph-specific Fab fragment (Fab315) in living cells
A mAb targeting histone H3 phosphorylated at Ser28 (H3S28ph) was adopted to evaluate the suitability of different fluorescent dyes for living cell imaging using dye-conjugated proteins. A series of research showed that both the chemical properties of the dyes and the linkers may influence Fab fragments function.
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Immunofluorescence and living cell localization of H3S28ph-specific Fab315 labeled with various dyes
The dyes of diverse wavelengths (around 488nm, 555nm, and 650nm) are conjugated to Fab315. It showed that some dyes can seriously affect Fab315.
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The effects on H3K9ac-specific Fab fragment (Fab310) in living cells led by different fluorescent dyes
The obtained results were similar to that of the above-mentioned experiments on Fab315.
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Photostability of different dyes in living cells
The photostability of different dyes varies so that a comparatively stable dye could be a better choice.
Conclusion
After a comprehensive evaluation of the suitability of fluorescent dyes as protein conjugation partner for living cell imaging, the team reached some preliminary conclusions.
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Green fluorescent dyes (with excitation at ~488 nm) affected the Fab binding affinity minimally, which might due to its fluorophore structure.
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ATTO488 could replace Alexa488 since it is more photostable.
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Red dyes have prominent photostability.
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Far-red dyes revealed lower contrast and high cytoplasmic spotty background.
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