Abstract
SummaryAdvanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.
Highlights
Labeling a protein of interest with an antibody is a well-established procedure in molecular biology
SUMMARY Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs)
We did not observe any co-diffusion of fluorescently labeled nanobodies with the green fluorescent protein (GFP)-labeled proteins
Summary
Labeling a protein of interest with an antibody is a well-established procedure in molecular biology. The popularity of antigen-binding fragments of antibodies and single-chain nanobodies derived from camelids or shark antibodies grew vastly (Beghein and Gettemans, 2017; Carrington et al, 2019; Leslie, 2018). Both types of molecules are much smaller than full-length antibodies, yet possess similar binding properties to their target proteins (Harmsen and De Haard, 2007; Sahl et al, 2017). The production methods and costs of generating a novel nanobody are higher than the ones for a standard monoclonal antibody; the nanobody can subsequently be produced and harvested from bacteria, yeast or mammalian cell culture and even recombinantly tagged (Arbabi Ghahroudi et al, 1997; Beghein and Gettemans, 2017; Pleiner et al, 2018)
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