A non-invasive way to detect vimentin cytoskeleton in live cells
Vimentin is the major intermediate filament in mesenchymal cells. In addition, vimentin has an important role as a biomarker for epithelial-mesenchymal transition (EMT). EMT is a highly dynamic cellular process involved in the initiation of metastasis and cancer. Vimentin is upregulated upon induction of EMT and is essential for cell migration and invasion.
To date there has been no approach available to study endogenous vimentin in a physiologic context. Overexpression of Vimentin fusion proteins induces EMT and therefore hinders any analysis. Now, with the new Vimentin-Chromobody, you can conduct a real-time analysis of endogenous vimentin in living cells. The Vimentin-Chromobody enables researchers to investigate the dynamic organization of vimentin intermediate filaments and to study EMT.
Live visualization with Vimentin Chromobody
With the Vimentin-Chromobody plasmid you obtain the sequence of the Alpaca antibody to vimentin fused to TagGFP (Evrogen). Upon transformation, your cells express the Vimentin-Chromobody and the vimentin filaments are visualized by binding of the fluorescent Chromobody. The binding does not influence cell viability or cell motility and thus offers you the unique possibility to non-invasively monitor vimentin cytoskeleton in your cells.
MDCK cells transfected with pVC-TagGFP2 plasmid (Vimentin Chromobody). The cells were fixed 48 h after transfection, cell nuclei were stained with DAPI (in blue). Vimentin- VHH-TagGFP highlights vimentin intermediate filaments in transfected cells (in green).
Chromobodies turn your cells into immuno-reporter factories
Nanobodies are unique, small antibody fragments that exhibit the extraordinarily efficacious binding of a targeted protein. Expressed in animal cells, Nanobodies can be collected and used for in vitro studies. Something unexpected happened when Nanobodies were expressed as a fusion to a reporter molecule such as GFP. The newly formed Chromobody bound directly to its target within the cells in which it was being made. Moreover, using the correct vector and promoters, the binding has both high specificity and low background. This provides an excellent alternative to GFP-fusions with the protein of interest for immuno-localization studies. Why? Because GFP- and RFP-fusions have been shown to block or alter the activity of the fused protein. In contrast, Chromobody act in trans as a reporter that will not interfere with the normal function of the targeted protein.
Camelidae single-domain antibodies are like IgGs on steroids
The family of animals known as Camelidae (camels, llamas, and alpacas) produce functional antibodies devoid of light chains, so-called "heavy chain" antibodies. These heavy chain antibodies recognize and bind their antigens via a single variable domain. When cleaved from their carboxy tail, these barrel-shaped structures (2x3 nm) are extraordinarily small, naturally-occurring, and intact antigen binding fragments (MW of 13 kDa). These fragments, called Nanobodies, are characterized by high specificity, affinities in the low nanomolar range, and dissociation constants in the sub-nanomolar range (typically 10- to 100-fold better than mouse IgGs). The compact size of Nanobodies makes them extremely stable at temperatures up to 70°C, and functional even in 2M NaCl or 0.5% SDS. These small and powerful antibody fragments can be used in a variety of unique applications. They will open up your research possibilities.
Vimentin Chromobody Immunodetection Vector
|Vector Type||Mammalian expression vector|
|Reporter||TagGFP2 (from Evrogen)|
|Transfection Method||Transfect mammalian cells by any known transfection method. If required, stable transformants can be selected using G418 [Gorman 1985]|
|Propagation in E. coli||DH5alpha, HB101, XL1-Blue, and other general purpose strains
Incompatible with pMB1/ColE1
|Selection||Prokaryotic – kanamycin
Eukaryotic – neomycin (G418)
|Replication||Prokaryotic – pUC ori
Eukaryotic – SV40 ori
|Use||Non-invasive live cell visualization of endogenous cytoskeleton or nucleus|