This method enables the production and application of bacterial expressed, functionalised nano-bodies to analyze retrograde protein transport from the cell surface to intercellular compartments.In particular the Trans-Golgi network.The main advantage of this technique is the use of sulfation.A highly specific, compartmentalized, and sensitive post-translation modification to assess reporter nano-body complex arrival in the DTN compartment.This method in combination with other derivatised nano-bodies for life cell imaging and electron microscopy can also be used to study endocitic processes of disease-related cell surface receptors.Our repertoire of functionalised nano-bodies has been engineered to study retrograde transport in cultured cells.However, these tools may also be applied in multicellular organisms.To begin this procedure, pick a bacterial colony containing the vector of interest from the plate and inoculate a flask containing 20 milliliters of LB medium supplemented with antibiotics.Incubate overnight at 37
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Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.
Buser, D. P., Spiess, M. Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells. J. Vis. Exp. (144), e59111, doi:10.3791/59111 (2019).