Transfection Reagent for Delivering Target Proteins into Cytosol ProteoCarry^™(Protein Transfection Reagent)

Going to Cytoplasm, not ending up in endosome!

This reagent introduces proteins and biological macromolecules (ex. dextran) into cells, especially cytosol. Conventional protein transfection reagents have been criticized for their retention in endosomes/lysosomes. However, this product can efficiently transport proteins into cytosol, and can be used in a wide range of experiments, such as evaluating the intracellular function of transfected proteins or inducing functional inhibition by introducing antibodies. In addition, unlike conventional reagents, this product does not require preincubation with proteins and does not form complexes, thus minimizing the impact on protein function.

※ The above number of assays is an estimate based on the protocol described in the datasheet. It varies depending on the experimental method and conditions.

  ☞ Product Information  

※ The transfection efficiency changes depending on various factors such as cell type, cell number, and protein type. The above information is just a guide, and it is recommended to optimize each experiment.

HeLa cells were treated with green fluorophore-conjugated dextran (Dextran-FL, average MW 10 kDa, final 200 μg/ml). When Dextran-FL alone was added to the cells (-), dot-like fluorescence signals were observed, but almost no fluorescence signal was observed in cytosol. On the other hand, when this product was used, fluorescence signals were observed throughout the cytosol, indicating that Dextran-FL was incorporated into the cytosol.

HeLa cells were treated with green fluorophore-conjugated IgG (IgG-FL, final 50 or 500 μg/ml). When IgG-FLs alone were added to the cells (-), they were taken up by endocytosis and dot-like fluorescence signals were observed, which were found to be retained in endosomes and lysosomes. On the other hand, when this product was used, even if dot-like endosome structures were observed, fluorescent signals from cytosol were also observed, and it was understood that IgG-FLs diffused from endosomes into the cells.
※ When observing fluorescence signals in cytosol, signals in cytosol are diluted because cytosol is larger than endosomes. If the amount of transducers is small, the fluorescence originating from endosomes may be markedly observed even if the transducers diffuse into cytosol. To detect the fluorescence signal in cytosol, it is recommended to add relatively high concentration of fluorescent-labeled proteins.

HeLa cells were treated with red fluorophore-conjugated anti-nucleus pore complex (NPC) antibody (anti-NPC-FL, final 50 μg/ml) in the presence or absence of ProteoCarry^™ (1x conc.) for 1 hour at 37oC. Without ProteoCarry^™ a little of fluorescent signal was observed in dot-like structure. On the other hand, ProteoCarry^™ clearly promoted localization of anti-NPC-FL to peri-nuclear structure. This result indicates that ProteoCarry^™ could induces incorporation of anti-NPC-FL into cytosol and binding of antibody to nucleus pore complex.

HeLa cells were treated with nuclear localization signal (NLS)-tagged EGFP (NLS-EGFP, final 10 μM) in the presence or absence of ProteoCarry^™ (1x conc.) for 1 hour at 37oC. Without ProteoCarry^™, a little of fluorescent signal was observed in endosome-like dot structures. On the other hand, ProteoCarry^™ promoted nuclear-localization of NLS-EGFP.

HeLa cells were transfected with a plasmid which encodes loxP-DsRed-stop-loxP-EGFP sequence by conventional lipofection reagent. Next day, the HeLa cells were treated with recombinant Cre recombinase (final 10 μM) for 1 hour at 37oC with or without ProteoCarry^™ (1x conc.). Without ProteoCarry^™, almost cells still keep DsRed expression under the treatment of Cre recombinase. On the other hand, ProteoCarry^™ promoted conversion from DsRed to EGFP by Cre/loxP recombination.

Saporin is an enzymatically cytotoxic protein which binds to ribosomes and inactivates ribosomes via its RNA glycosidase activity. RAW264.7 cells were treated with saporin (final 10 μg/ml) with or without ProteoCarry^™ for 1 hour at 37oC. After 24 hours culture, cell viability was measured by MTT assay. Without ProteoCarry^™, saporin induced cell death with less than 30% efficiency. On the other hand, ProteoCarry^™ dramatically increased cytotoxicity of saporin and almost cells were dead (>90%).

To monitor intracellular localization of ProteoCarry^™, fluorophore-labeled ProteoCarry^™ (ProteoCarry^™-FL) was prepared and HeLa cells were treated with ProteoCarry^™-FL. Fluorescent signal was observed by confocal microscopy. Signal was detected mainly in endosomal dot like structure and little fluorescent signal was observed in cytosol. This data indicates ProteoCarry^™ keeps binding to endosomal membrane after lysis of endosomes.